EVALUATION OF PROCEDURES FOR PREPARING ENVIRONMENTAL
AND WASTE SAMPLES FOR MUTAGENICITY TESTING:
Environmental Waters and Wastewaters
by
Y.Y. Wang, C.P. Flessel, M.J. DiBartolomeis, Jr.,
K. Chang, and S. Sun
Air and Industrial Hygiene Laboratory Section
California State Department of Health Services
Berkeley, California 94704-9980
Cooperative Agreement CR810022-02-0
Final Report
Project Officer
Llewellyn R. Williams
United States Environmental Protection Laboratory
Environmental Monitoring Systems Laboratory
P. 0. Box 15027
Las Vegas, Nevada 89114
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
LAS VEGAS, NEVADA 89114
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NOTICE
The information in this document has been funded wholly or in part by
the United States Environmental Protection Agency. This document is
intended for internal Agency use only. Mention of trade names or
commercial products does not constitute endorsement or recommendation
for use.
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ABSTRACT
To complement the standardized protocol of the EPA for conducting the Ames
assay, consensus protocol for the preparation of environmental samples for testing
are currently under evaluation. The first of these is for environmental water and
wastewater.
The Salmonella/mammalian-enzyme assay developed by Dr. B.N. Ames and his
colleagues has been widely accepted as the most frequently-used and reliable short-
term mutagenicity assay available. It is one of the most cost-effective screening
tools for evaluating the distribution of mutagenic pollutants in the environment. The
application of the Ames assay for mutagenicity measurement can also be valuable
for prioritizing environmental samples for chemical analysis, for facilitating the
detection of potentially hazardous environmental contaminants, and eventually, for
assessing risks of human and environmental exposure.
However, most complex environmental samples can not be tested directly in
the Ames assay due to the presence of very low concentrations of mutagens or the
existence of toxic components which mask mutagenic effects. Protocols are needed
for preparation of environmental and waste samples for mutagenicity testing. Under
the sponsorship of the EPA, a project to develop guidelines for sample preparation
of various environmental media including wastewater, drinking water, soils and
sediments, solid wastes, air, and nonaqueous liquid wastes has been initiated.
Standardization procedures are crucial for ensuring comparability of test results for
scientific evaluation and for potential enforcement and litigation actions arising from
surveillance of hazardous waste sites.
The first protocol evaluated is for environmental water and wastewater.
Samples used in the evaluation were selected from a range of generic wastewater
types and include effluents from industrial and municipal wastewater treatment
plants, contaminated groundwater, surface runoff from a hazardous waste landfill,
the aqueous fraction from a RCRA solid waste extraction procedure, and brackish
surface water receiving industrial effluents and areal runoff. The aqueous wastes
were processed by a liquid-liquid extraction scheme similar to the EPA method 625.
A 3 L sample was typically used for the extraction. The sample was adjusted to
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pH 11, extracted with dichloromethane, then adjusted to pH 2 and reextracted with
dichloromethane. The dichloromethane fractions were either combined or were kept
separated, then concentrated to dryness. The residues of the solvent extracts were
tested in the Ames assay using strains TA98 and TA100 in the absence and the
presence of 2%, 10%, or 30% rat liver activation systems (S-9). All the field samples
were found to be mutagenic, and mutagen levels ranged from approximately 200 to
4000 revertants per liter of water. TA98 was the more sensitive strain although
mutagenic activity was also detected in TA100 for some samples.
ASTM Type I water, which served as field and travel blanks as well as method
blank, was extracted, and the residue was tested in the Ames assay. It was established
as the method background by using several statistical analyses. Control charts for
precision and accuracy of the background were constructed for routine quality control
checks on data acceptability. The average water blank values in TA98 were 35
(without S-9), 49 (2% S-9), 51 (10% S-9), and 39 (30% S-9) revertants/plate. The
detection limit was set as twice that of the background according to the Ames
two-fold rule.
Replicate or triplicate measurements of mutagenicity were performed on the
wastewater samples. The results of several samples with relatively stable activities
were evaluated for precision of the experimental process. Standard deviations
representing the precision of these measurements varied from 2% to 33%. The
accuracy of the methodology was evaluated indirectly through recovery studies. The
extraction efficiency based on the recovery of spiked mutagens was determined with
both chemical and mutagenicity analyses. The recovery for benzo(a)pyrene, a neutral
compound, was 90% to greater than 100% in a municipal wastewater sample. For
4-nitrobenzoic acid, an acidic compound, the recovery was 40% to 60%. The recovery
of 2-aminoanthracene, a basic compound, was approximately 25% to 35%.
The study established the validity of the consensus protocol for the preparation
of a variety of generic types of wastewater samples for Ames testing. Emphasis
was on minimum requirements for routine screening of wastewater samples, consistent
with the intended purpose of the sample preparation protocol for eventual use in
hazardous materials monitoring.
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This report covers a period from March 1, 1985, to December 31, 1985, and
work (including draft report preparation) was completed as of March 31, 1986.
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CONTENTS
Notice ii
Abstract Hi
Figures viii
Tables ix
Abbreviations. x
Acknowledgements xi
1. Introduction 1
Goals and Tasks 2
Approach 4
2. Conclusions 5
3. Recommendations 7
Topics for Further Evaluation of the Wastewater Protocol .... 7
XAD-Resin Method 7
Sample Degradation 8
Extraction Efficiency 8
Quality Assurance (QA) Plan 9
4. Materials and Methods 10
Apparatus, Equipment, and Methods 10
Consensus Protocol for Environmental Waters and Wastewater. . . 11
5. Experimental Procedures - Field and Laboratory Applications 14
Six Genenc Sample Types 14
Sample Collection, Log-In, and Storage 19
Phase Separation 20
Liquid-Liquid Extraction 24
Ames Assay 27
Assay Scheme 27
Quality Control (QC) of the Ames Assay 29
Extraction Efficiency 31
Chemical Analysis by HPLC 32
Mutagenicity Recovery Analysis 32
6. Results and Discussions 35
Wastewater Protocol Evaluation 35
Ames Assay Results and Interpretation 35
Surface Runoff from a Class I Landfill 37
Brackish Surface Water Receiving Industrial Effluents . 37
Municipal Wastewater Treatment Plant Effluent ... 42
Industrial Wastewater Treatment Plant Effluent ... 42
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CONTENTS (continued)
Contaminated Groundwater from the Stringfellow
Hazardous Waste Disposal Facility 45
EPA/NBS Reference Sludge-TCLP Leachate ... 48
Possible Changes in Sample Composition .... 53
Liquid-Liquid Extraction 56
Samples Form Emulsions 56
Samples Form Precipitates 59
Residual Water 60
Replace Kudema-Danish Apparatus with
Rotary Evaporator 60
Mutagen Extraction Efficiency 61
Development of QA Program 65
General Goal and Specific Objectives 66
QC Approach 68
Statistical QC Analysis and Records 69
Method Background 69
QC Charts for Accuracy and Precision 71
References 78
Appendices 84
A. Original Protocol for Environmental Waters and Wastewater .... A-84
B. Original Consensus Protocol for Drinking Water B-95
C. Sample Collection Information Record C-116
D. Primary Ames Bioassay Data and Dose-Response Curves for the
Protocol Validation Study D-125
E. Primary Ames Bioassay Data for the Recovery Study E-250
F. Strain Finction, Cell Titer, and Viability Record F-303
G. Primary Data Work Sheet for Statistical Analysis G-317
H. Primary Data Work Sheet for QC Chart Calibration H-321
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FIGURES
Number Page
1. Wastewater Sample Process Flow Diagram 12
2. Sample Processing Scheme 23
3. Liquid-Liquid Extraction Scheme 25
4. Ames Assay Scheme 28
5. HPLC Chromatograph of Spiked Reference Mutagens 34
6. Dose-Response Curves Showing Degradation of Mutagens in a 55
Stringfellow Contaminated Groundwater Sample
7. Dose-Response Curves Showing Degradation of Mutagens in a Surface 57
Runoff Sample from a Class I Hazardous Waste Landfill
8. Dose-Response Curves for a Municipal Wastewater Treatment Plant 58
Effluent Sample with Stable Mutagenicity
9. Control Charts of the Method Background in TA98 without S-9 73
10. Control Charts of the Method Background in TA98 with 2% S-9 Mix 74
11. Control Charts of the Method Background in TA98 with 10% S-9 Mix 75
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TABLES
Number Page
1. Mutagenicity of Wastewater Samples Prepared by the 6
Liquid-Liquid Extraction Method in Strain TA98
2. Recommended Volumes and Storage of Samples 21
3. HPLC Gradient Conditions for the Separation 33
of Spiked Reference Mutagens
4. Wastewater Sample Description and Characterization 37
5. Mutagenicity of a Surface Runoff Sample from a Class I Landfill, 39
Sample No. AIHL-85-0405
6. Mutagenicity of a Brackish San Francisco Bay Surface Water Sample, 40
Sample No. AIHL-85-0404
7. Mutagenicity of a Brackish Receiving Surface Wastewater Sample 41
from the Discharge Site of an Industrial Wastewater Treatment Plant,
Sample No. AJHL-85-044A
8. Mutagenicity of a Municipal Wastewater Treatment Plant 43
Effluent, Sample No. AIH_-85-0403
9. Mutagenicity of an Industrial Wastewater Treatment Plant 44
Effluent, Sample No. AIHL-85-0406
10. Mutagenicity of a Contaminated Groundwater from the Stringfellow 46
Hazardous Waste Disposal Facility On-Site Well, OW-2,
Sample No. AIHL-85-0402
11. Mutagenicity of a Contaminated Groundwater from the Stringfellow 49
Hazardous Waste Disposal Facility Upgradient Well, UGB-8,
Sample No. AIHL-85-042A
12. Mutagenicity of an EPA/NBS Reference Sludge-TCLP Leachate, 50
Sample No. AIHL-85-0401
13. Investigation of the Mutagenicity of Sodium Acetate Buffer 52
Used for the TCLP Leachate Preparation
14. Mutagenicity of Chemicals Identified in the TCLP Leachate Sample 54
15. Liquid-Liquid Extraction Recovery of Three SPRM's B(a)P, and 2AA 63
and 4NBA Added to the Laboratory Distilled Water
16. Liquid-Liquid Extraction Recovery of Three SPRM's, B(a)P, 2AA and 64
4NBA Added to the Municipal Wastewater Effluent
Sample (No. AIHL-85-0403)
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ACS:
AIHL:
ASTM:
2AA:
B(a)P:
CDHS:
CDHF:
CFR:
CWQCB:
DMSO:
EP:
EPA:
EMIC:
EMSL:
FIFRA:
HML:
HPLC:
K-D:
4NBA:
NBS:
2NF:
4NQO:
QA:
QC:
RCRA:
SAIC:
SD:
SPRM:
SR:
TCLP:
TSCA:
T5CD:
ABBREVIATIONS
American Chemical Society
Air and Industrial Hygiene Laboratory, CDHS
American Society for Testing and Materials
2-Aminoanthracene
Benzo(a)pyrene
California State Department of Health Services
California Public Health Foundation
Code of Federal Regulations
California State Water Quality Control Board
Dimethylsulfoxide
Extraction Procedure (40 CFR 261.24)
Environmental Protection Agency
Environmental Mutagen, Carcinogen, and Teratogen Information Center,
Oak Ridge, TN
Environmental Monitoring System Laboratory, EPA, Las Vegas, NV
The Federal Insecticide, Fungicide and Rodenticide Act
Hazardous Materials Laboratory, CDHS
High Pressure Liquid Chromatography
Kuderna-Danish Concentrator
4-Nitrobenzoic Acid
National Bureau of Standards
2-Nitrofluorene
4-Nitroquinoline-N-Oxide
Quality Assurance
Quality Control
The Resource Conservation and Recovery Act
Science Applications International Corporation, La Jolla, CA
Standard Deviation
Spiked Reference Mutagens
Spontaneous Mutation Revertants
Toxicity Characteristics Leachate Procedure
The Toxic Substances Control Act
The Toxic Substances Control Division, CDHS
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ACKNOWLEDGEMENTS
The authors acknowledge the following collaborators for their contribution to the
success of this project:
Mark Galloway, T5CD, CDHS, Sacramento, CA; for making arrangements to collect
the Stringfellow samples.
Wilson Horn, SAIC, La Jolla, CA; for collecting the two Stringfellow groundwater
samples.
Tom Li, John Hennings, Mi lad Iskander, Howard Okamoto, and Jane Tang, HML,
CDHS, Berkeley, CA; for analyzing the Stringfellow contaminated groundwater
sample (No. AIH-85-0402) for organic chemicals and metals.
Kai-Shen Liu, AIHL, CDHS; for advice on statistical analyses.
Barton Simmons, HML; for arranging the abovementioned analyses, for providing
liaison between agencies, and for overall consultations to the project.
Harold J. Singer, CWQCB, Oakland, CA; for collecting the municipal (No. AIHL-85-
0403) and industrial (No. AIHL-85-0406) wastewater samples and the runoff
sample (No. AIHL-85-0405); and for consultations on sample selection.
Robert D. Stephens, Chief of HML; for general support to the project.
Jerome J. Wesolowski, Chief of AIHL; for overseeing the project, for providing liaison
between CDHS and CPHF, and for general support and consultations.
We also thank Robert C. Stafford, EMIC, Oak Ridge National Laboratory, Oak Ridge,
TN, for computer literature searches on mutagenicity of chemicals. Especially, we
thank Helen K. Lu, David Ting, and Vincent Wong, CPHF, for their industrious
technical assistance. The word processing assistance of Carol A. Chin, CDHS, is
greatly appreciated. Finally, special appreciation is expressed to Llewellyn R.
Williams, EMSL, EPA, Las Vegas, NV for research contributions and for support.
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SECTION 1
INTRODUCTION
There is increasing evidence that environmental mutagens are a cause of
cancer and of genetic birth defects and that they may also contribute to aging and
heart disease (Ames, 1979). Of particular concern are toxic and hazardous chemical
wastes which are produced in quantities of over 250 million metric tons a year in
the United States. The U.S. Environmental Protection Agency (EPA) has found that
the mismanagement of these wastes causes environmental and public health damage
such as contamination of groundwater and surface waters, pollution of air and soils,
and poisoning and chronic illness of humans and animals via the food chains or direct
contact (USEPA, 1979a). The chemical complexity of hazardous wastes and their
residues precludes complete chemical analysis of toxic components. Furthermore,
toxicological synergism and antagonism between chemical waste components is likely.
These interactions make risk assessment of hazardous wastes on a chemical-by-
chemical basis extremely difficult. A critical need is to develop short-term biological
methods to assist in assessing the potential hazards of chemicals in complex waste
samples. To this end, EPA laboratories and program offices with responsibility for
toxic and hazardous substances management have expressed an immediate need to
apply the Ames and other mutagenicity tests in the analysis of complex waste
samples. Short-term bioassays, such as the Ames test, are now used by many public
and private laboratories in screening of complex mixtures for mutagenic activity
(Hollstein and McCann, 1979). Results of these tests are often widely circulated
and interpreted. It is thus critical that testing procedures be standardized and that
the quality of the data be assured so that valid interlaboratory and intralaboratory
comparisons of results obtained by different laboratories can be obtained.
This report describes progress in developing guidelines for preparing environ-
mental waste samples for mutagenicity (Ames) testing. Procedures for the preparation
and the mutagenicity testing of environmental waters and wastewater are described.
This work was carried out under a cooperative agreement (CR810022-02-0) between
the Environmental Monitoring Systems Laboratory (EMSL), EPA, Las Vegas, Nevada,
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and the Air and Industrial Hygiene Laboratory (AIHL), California State Department
of Health Services (CDHS), administered through the California Public Health
Foundation (CPHF) during the period from March 1, 1985 to December 31, 1985.
GOALS AND TASKS
The ultimate objective of this work is to develop a quality assurance (QA)
program for biological testing of complex environmental samples using the Ames
Salmonella mutagenicity assay. Mutagenicity testing can be used to determine the
mutagenic potential of complex environmental pollution mixtures. In preparing
mixtures for Ames testing, it is critical to evaluate differences in capability and
efficiency of sample preparation procedures toward organic compounds that might
not be detected by chemical analyses alone. Such knowledge will increase the value
of the Ames test to EPA hazardous waste monitoring programs and to other programs
assessing environmental and human health risks.
The Salmonella/mammalian-enzyme mutagenicity assay developed by Ames and
his colleagues (Ames et al., 1975; Maron and Ames, 1983) has been extensively
studied and widely accepted as the most reliable and efficient short-term mutagenicity
assay available (Brusick and Young, 1981; Hollstein and McCann, 1979, Sexton et al.,
1981). In a collaborative study supported by the EPA, a standard Ames test protocol
has been established and validated (Adams et al., 1984; Williams and Preston, 1983,
Williams, 1985). The utility of this procedure as a means of providing routine
detection of potentially mutagenic substances in the environment has also been
recommended (Sugimura and Nagao, 1982).
However, the interim procedures of the EPA for conducting the Ames test
(Williams and Preston, 1983) do not address the sample preparation of environmental
wastes and substances. Further, there are no generally accepted or standard methods
specifically designed for "preparing these types of samples for biological testing. The
EPA requires standardized procedures for the preparation of environmental samples
for mutagenicity testing to ensure comparability of test results for scientific
evaluation and for potential enforcement and litigation actions arising from
surveillance of hazardous waste sites.
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The purpose of this phase of the project is to validate guidelines for preparing
environmental water and wastewater samples for the Ames test. The guidelines are
based on a consensus protocol for wastewater sample preparation developed under
the auspices of the Quality Assurance Division, EMSL-Las Vegas, EPA (ICAIR, 1985).
At present, preliminary protocols for the following six media are available: air
particulate matter, drinking water, environmental waters and wastewater, nonaqueous
liquid wastes, soils and sediments, and solid wastes. However, these protocols have
not been validated, and no guidance for QA exists in these protocols. Therefore
the main tasks of this project are protocol validation and QA development. In this
report, guidelines for preparing wastewater are developed. Guidelines to the other
five media will be developed in future phases of the work.
The goal of the sample preparation protocol is to provide samples which
accurately and reliably reflect the mutagenic potential of the original complex
material. These procedures should also yield adequate products that can be
appropriately tested in the Ames assay and to other biological assay systems. In
addition, since the protocols are intended for routine use in the examination of a
large number of samples, the procedures need to be simple and cost-effective. The
extent of sample preparation processing required varies for different media. For
example, nonaqueous liquid waste samples in many cases can be tested directly for
mutagenic activity without any sample preparation. However, extraction and
concentration are required for the other five media: air particulate matter, drinking
water, wastewater, soils and sediments, and solid wastes (ICAIR, 1985). In general,
further fractionation of an extract is required if cytotoxicity of the extract prevents
assessment of its mutagenic potential. By using standardized and validated procedures
for sample preparation and for mutagenicity testing, it will be possible to increase
the accuracy and the precision of the results and to permit more meaningful
interlaboratory comparisons.
Another essential aspect of the validation of the wastewater preparation
protocol includes the development of a comprehensive QA program incorporating the
protocol. This protocol may also be used for preparing wastewater samples for other
bioassay systems and for chemical analyses. Once validated, these standardized
testing procedures can be applied to assist in the wastewater monitoring effort of
the EPA.
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APPROACH
The development of a comprehensive QA program for mutagenicity testing of
environmental mixtures using the Ames test involves three phases. In the first
phase, a standard Ames test protocol is established and is validated through inter-
laboratory testing. This is now completed (Adams et al., 1984; Williams and Preston,
1983). In the second phase, standard protocols for the preparation of complex
environmental mixtures for Ames testing are developed and validated. In the third
phase, the validated protocols for sample preparation and mutagenicity testing of
complex mixtures are applied to actual waste samples by laboratories in support of
EPA hazardous waste and other programs. In this stepwise fashion, mutagenicity
testing procedures can be standardized and can be applied to the monitoring of
hazardous waste materials by EPA.
This Cooperative Agreement is concerned with phase two, the evaluation of
sample preparation protocols. Specifically, the consensus sample preparation protocol
for wastewater is validated during this reporting period. Although the application
phase is beyond the scope of the current EPA-CDHS/CPHF" Cooperative Agreement,
actual waste samples are used in developing the standardized sample preparation
protocol. This provides preliminary indications as to the efficacy of using the Ames
assay and sample preparation protocols as a screening mechanism for potentially
hazardous environmental water and wastewater samples.
The following four-step approach is used in the protocol evaluation and
validation:
- Establish the method background with field blanks and with reagent blanks. Several
quality control (QC) procedures to avoid potential problems such as chemical
contamination, i.e., artifact mutagen formation and negative interference, are
developed.
- Establish the accuracy of the method through the use of spikes (analyte addition)
or surrogates (chemical homologues) or both. To assess accuracy for the sample
collection process, travel blanks are carried along with the field samples during
collection and transport.
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- Establish the precision of the method through testing of replicate aliquots of the
same sample in a batch. The precision is affected by the homogeneity and the
stability of the sample as well as the extraction and the analytical techniques of
the individual analyst.
- Establish the best available protocols through method comparison for recovery
efficiency and for cost-effectiveness with an emphasis on the minimum requirements
for the purpose of routine screening.
Two major tasks, the validation of the wastewater protocol and the development
of a QA program, have been completed during the period of time from March 1,
19B5, to December 31, 1985. The progress on technical design, procedure documen-
tation, experimental method and results, statistical analyses, and precision and
accuracy establishment are presented in this report.
SECTION 2
CONCLUSIONS
Based on the data gathered so far, it is concluded that the EPA-consensus
liquid-liquid extraction protocol (Appendix A), as modified herein, can provide extracts
suitable for the Ames testing from the six types of wastewater samples (Table 1).
As long as the sample material is available and the sample stream is not constantly
changing over time, the liquid-liquid extraction can be cost-effective. Standard
deviation (SD), used for precision evaluation, was 9% for the industrial effluent (No.
AIHL-85-0406), 20% for the municipal effluent (No. AIHL-85-0403), 2% for the
brackish receiving water (No. AIHL-85-044A), and 33% for the bay water (No.
AIHL-85-0404). The recovery of spiked reference mutagens (SPRM's) ranged from
25% to 35% for 2-aminoanthracene (2AA), 40% to 60% for 4-nitrobenzoic acid (4NBA),
to 90% to 105% for benzo(a)pyrene (B(a)P). The method background was established
using the water blank value which was 35 (without S-9), 49 (2% S-9), 51 (10% S-9),
and 39 (30% S-9) revertants/plate in TA98.
Three major problems occurred during the interference by extraction process:
(1) precipitation at pH 11, (2) formation of emulsions, and (3) interference by residual
water. Modifications to improve the extraction procedure are: (1) extracting the
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TABLE 1. MUTAGENICITY OF WASTEWATER SAMPLES PREPARED BY THE LIQUID-
LIQUID EXTRACTION METHOD IN STRAIN TA98
Sample (No. AIHL-85-)
S-9
Condition
Mutagenic Activity + SD*
(Revertants/L)
Stringfellow Groundwater
(on-site well, OW-2)
(0402)
2%
36002
NBS Reference Sludge
- TCLP Leachate
(0401)
2%
23003
Industrial Treatment
Plant Effluent
(0406)
30%
1700 + 1604
Landfill Surface Runoff
(0405)
without
2
610
Municipal Treatment
Plant Effluent
(0403)
10%
420 +, 80
Brackish Receiving Wastewater
(044A)
2%
240 ± 6
Brackish S.F. Bay Water
(0404)
2%
230 + 70
Stringfellow Groundwater
(upgradient well, UCB-8)
(042A)
2%
1705
1. Standard deviation (SD) representing precision measurements was obtained from
results or replicate or triplicate experiments.
2. No replicate result was obtained as a result of possible sample degradation.
3. Only one acceptable result was produced, see Section 6.1.1.6, EPA/NBS Reference
Sludge - TCLP Leachate, for details.
4. Data produced approximately four months after sample collection. See Section
6.1.1.4, Industrial Wastewater Treatment Plant Effluent, for details.
5. Only one experiment was performed for this sample, see Section 6.1.1.5,
Contaminated Groundwater from the Stringfellow Hazardous Waste Disposal
Facility, for details.
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samples which precipitate at pH 11, at pH 2 first; (2) eliminating the emulsion with
glass wool, and (3) absorbing the residual water with a sodium sulfate column.
In addition to the sample preparation procedures, a sequential Ames
mutagenicity testing strategy developed in this project proved to be efficient in cost
and effort. A screening experiment is performed at first, and only the optimum
condition is repeated. Only the two most sensitive strains, TA98 and TA100, for
environmental complex mixtures are used. Four conditions of S-9 (without, with 2%,
10%, and 30% S-9 mix) are applied strategically and cover a wide spectrum of
activating enzyme requirements for a large number of chemicals. Sequential testing
improves efficiency by eliminating the need for further large scale experiments once
a sample has been found to be mutagenic, and the optimum testing condition has
been established.
SECTION 3
RECOMMENDATIONS
TOPICS FOR FURTHER EVALUATION IN THE PREPARATION OF WASTEWATER
SAMPLES
XAD-Resin Method
As described in Section 6.1.1.4, Industrial Wastewater Treatment Plant Effluent,
the mutagenic activity initially was not detected in extracts from the industrial
waste sample (No. AIHL-85-0406). The extracts tested were obtained by extraction
of up to 3 L of sample with dichloromethane. One of the limitations of the
liquid-liquid extraction method is that it can not be used to concentrate large
samples. When mutagenic activity is below detection using liquid-liquid extraction
of 3 L samples, it is useful to process larger quantities of material. In principal,
this can be accomplished using sorbant resin concentration according to the EPA-
consensus drinking water protocol (Appendix B). Several preliminary experiments
were carried out applying XAD-2 and XAD-7 resin columns to concentrate larger
volumes (10 L) of several samples. However, these pilot experiments did not always
yield larger amounts of organics from the 10 L sample comparing with the liquid-liquid
extraction of the 3 L sample. Furthermore, these extracts were either nonmutagenic
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or toxic to the tester bacteria. Thus, extracting 10 L samples using the XAD method
did not exhibit higher total mutagenic activity compared to the liquid-liquid extraction
of 3 L samples. Future work is required to explore the problems of residue toxicity
and of recovery in wastewater extracts isolated from XAD resin columns. The
liquid-liquid extraction procedure and the XAD method should be compared for the
preferential concentrating mutagens versus toxicants.
Sample Degradation
Among the six types of samples evaluated with the liquid-liquid extraction
method, two. samples exhibited significant decreases in mutagenicity over a period
of time (See Section 6.1.1.7, Possible Changes in Sample Composition, for details):
the surface runoff from a class I landfill (See Section 6.1.1.1, Surface Runoff from
a Class I Landfill, for details) and the contaminated groundwater from the Stringfellow
Hazardous Waste Disposal Facility (See Section 6.1.1.5, Contaminated Groundwater
from the Stringfellow Hazardous Waste Disposal Facility, for details). These samples
were highly mutagenic in the initial testing. After only approximately two to three
weeks of storage at 4°C in the dark, the activity was reduced by more than 50%.
The cause for these decreases is not known. It may be caused by combinations
of chemical reactions, by microbial activities, or by storage conditions. A time-course
study on several representative samples is also needed to evaluate the nature and
extent of mutagen degradation. This will provide necessary information for
determining the maximum sample storage time and the optimum storage conditions.
Extraction Efficiency
Three mutagens, 4NBA, B(a)P, 2AA, were used for evaluating the extraction
efficiency of the liquid-liquid extraction procedure (See Section 6.1.3, Mutagen
Extraction Efficiency, for details). The mutagen 4NBA is an acidic compound; B(a)P,
a neutral compound; and 2AA, a basic compound. The recovery efficiencies of these
chemicals spiked in distilled water varied from 60% to 80% for 4NBA to 80% to
greater than 100% for B(a)P and 2AA. Similar recovery was obtained in the municipal
wastewater sample (No. AIHL-85-0403) spiked with B(a)P. For 4NBA, the recovery
was 40% to 60% in the wastewater sample. The recovery of 2AA was substantially
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reduced to 25% to 35% in the mmicipal wastewater. Several possibilities may have
contributed to the lower extraction efficiencies including chemical reactions or lasses
as a result of emulsion formation. These possibilities should be investigated further,
and evaluation with other known mutagens is needed to verify the overall performance
of the extraction method.
QUALITY ASSURANCE (QA) PLAN
In addition to the modifications in the sample preparation procedures suggested
above, a number of steps are also recommended for the establishment of a QA
program for sample preparation and mutagenicity testing of environmental wastewater
samples.
The establishment of a QA plan makes possible the collection of consistent
data through the use of systematic methods. QA represents the total integrated
program for assuring the reliability of monitoring and measurement data (Booth,
1979). The assurance of this reliability is, in turn, maintained through the use of
discrete quality control (QC) activities such as the chain-of-custody documentation
of sample handling, the routine calibration of instruments, the purity and cleanness
assurance of consumables and glassware, the establishment of method background,
the construction of QC charts for precision and accuracy, the routine analyses of
SPRM's, the monitoring of positive and negative controls, and the application of
strain function check procedures for obtaining prescribed standards of performance.
There are several QA procedures that allow the project manager(s) to judge
and to monitor the quality of the procedures performed by the analytical staff. The
first of these procedures is planning the QA program. This planning covers all
aspects of acquiring quality data. In general, it is essential that a QA project plan
be written and approved before the initiation of sampling and of laboratory analysis.
The plan should cover but not be limited to the following areas:
- Project description, objectives, and policy.
- Project organization and responsibilities.
- QA management.
- Personnel qualification, facilities, equipment, and services.
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- QA objectives in terms of precision, accuracy, completeness, representativeness,
and comparability.
- Sampling and analysis procedures assuring that only appropriate methods are used.
- Sample custody.
- Calibration procedures.
- Data generation and processing.
- Internal quality control checks.
- Data analysis, validation, and reporting.
- Data quality assessment.
- Performance and system audits.
- Preventive maintenance.
- Specific procedures to be used to routinely assess data precision, accuracy, and
completeness.
- Corrective action.
- Quality assurance reports to management.
A second recommended QA method is the use of periodic on-site inspection
and QA audits by the project manager. These activities are for the purpose of
verifying that the QC procedures listed on the checklists are being performed and
that they are sufficient. These inspections are performed for sampling, analysis,
and data management activities.
SECTION 4
MATERIALS AND METHODS
APPARATUS, EQUIPMENT AND MATERIALS
The validation of the sample preparation protocol for environmental water
and for wastewater involves both the wastewater protocol and the drinking water
protocol. A complete copy of these two consensus protocols is attached as Appendix
A and Appendix B. The apparatus and equipment listed on the original consensus
protocols were applied in experiments. In addition, the following items were also
used:
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- ASTM Type I water prepared with a MILLI-Q® Water Purification System with
three cartridges: ion-exchange, carbon, and prefilter; and a TWIN-90 filter unit
(MILLIPORE CORPORATION, Bedford, Massachusetts) or equivalent. The American
Society for Testing and Materials (ASTM) specifies four different grades of water
for use in methods of chemical analysis and of physical testing. The ASTM Type
I grade water is, by definition, prepared by the distillation of feed water having
a maximum conductivity of 20 ymho/cm at 25°C followed by polishing with a
mixed bed of ion-exchange materials and a 0.2-ym membrane filter. At AIHL,
this water is obtained with a MILLIPORE MILLI-Q® water purification system.
- pH meter, CORNING Model 10 or equivalent.
- Standard buffer solutions for pH meter calibration, pHydrion Buffers (MICRO
ESSENTIAL LABORATORY, Brooklyn, New York) or equivalent.
- Rotary evaporator, BUCHI Rotavapor R110 or equivalent.
- Reference mutagen standards. In this project, the chemicals were provided by the
EPA Chemical Repository through Dr. Llewellyn R. Williams, EPA, LV.
- The equipment and materials required for the Ames bioassay (Ames et al., 1975;
Maron and Ames, 1983) were applied as specified in the EPA interim procedures
(Williams and Preston, 1983).
- Reagents used for preparing the acetate buffer for TCLP extraction were all ACS
(American Chemical Society) reagent grade: anhydrous sodium acetate, glacial
acetic acid, and sodium hydroxide.
CONSENSUS PROTOCOL FOR ENVIRONMENTAL WATERS AND WASTEWATER
The consensus wastewater sample preparation protocol (See Appendix A) being
validated in this project is summarized in the flow chart as shown on Figure 1.
Wastewater samples may contain solids, nonaqueous liquids, and aqueous liquids. Each
sample container should be stored in the dark, motionless at 4°C for a minimum of
24 hours after receipt. After the settling period, different phases are separated
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Collected Sample
\
24—h Gravity Separation
Nonaqueous Liquid
Phase
Aqueous Liquid
Phase
Veigh and Determine
Volume and Percent
Suspended Solids
/ \
Solid Sediment
Phase
Ca) <5Z Solids
by Weight
Process by Nonaqueous
Liquid Wastes Protocol
>52 Solids
by Weight
(b) Liquid-Liquid
Extraction
(c) Filter
or Centri-
fuge Sample
to Separate
Liquid from
Suspended
Solids
Bioassay
Process by
Drinking
Water
Protocol
Bioassay
Concentrate
~
Solvent
Exchange
I
Bioassay
/ \
Process by
Waste Solids
Protocol
Bioassav
Liquid Solids
Process
by Drinking
Water
Protocol
Bioassay
Process
by Waste
Protocol
Bioassay
Note: If results of (b) are negative (not mutagenic), it is recommended that
the sample be subjected to (a) or (c) processing and that it be retested
in the bioassay.
Figure 1. Wastewater sample processing flow diagram.
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according to gravity. Any nonaqueous liquid phases identified tn the liquid component
of the sample are separated from the containers, are combined into one sample, and
are processed as a nonaqueous liquid waste. Any solid sediment on the bottom of
the sample container is collected, is combined into a single sample, and is processed
as a solid waste. Each liquid or solid phase recovered must be weighed, and the
volume must be determined before further processing. Like phases are combined
into a common vessel for processing. Once combined, total weights and volumes
are calculated. Storage conditions are the same as defined for the initial field
sample.
The aqueous liquid phase recovered from the sample is processed by one of
the following methods:
a. If the sample has < 5% suspended solids by weight (EPA Method 160.2, in
USEPA, 1979b), the sample may be extracted and concentrated by the
techniques described in the Drinking Water Protocol (XAD resin chromato-
graphy, see Appendix B for a complete copy) with the addition of a celite
prefilter column to the concentrator apparatus.
b. If the sample has > 5% suspended solids, it may be processed by a liquid-liquid
extraction method using a 3 L 3ample. If the bioassay result of the sample
recovered from this method is negative, consideration should be given to
processing a retained liquid phase (10 L) by XAD resin chromatography as
described in the Drinking Water Protocol.
c. As an alternative to b., if the sample has > 5% suspended solids by weight,
the sample may be further separated by high-pressure filtration or by high-speed
centrifugation into liquid (< 5% suspended solids) and solids. These two phases
can be processed further by the Drinking Water Protocol and by the Waste
Solids Protocol, respectively.
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SECTION 5
EXPERIMENTAL PROCEDURES - HELD AND LABORATORY APPLICATIONS
The wastewater protocol validation process involves three aspects. First, the
applicability of the protocol for providing sample extracts suitable for the Ames
assay system is evaluated using various generic types of wastewater samples. Second,
the adequacy of the protocol for extracting compounds representative of the original
sample is studied by determining the extraction efficiency of SPRM's. Third, the
validity of the protocol in avoiding the artificial generation of mutagens is established
by developing the method background levels with blank water controls and by
comparing these with the solvent and spontaneous mutation background in the Ames
test.
SIX GENERIC SAMPLE TYPES
Since environmental water and wastewater may contain samples with a wide
range of chemical constituents with different reactivities and extraction efficiencies,
samples were collected which included most generic types of wastewater. The
purpose is to prove that the protocol provides suitable extracts for the Ames testing
from various types of wastewater samples.
Six types of wastewater samples were collected. These samples were the
following:
- effluent from a municipal wastewater treatment plant;
- effluent from an industrial wastewater treatment plant;
- surface runoff from a hazardous waste landfill;
- aqueous leachate from solid waste extraction procedures;
- brackish estaurine surface water receiving industrial effluents and areal runoff;
- contaminated groundwater.
In collaboration with the California State Water Quality Control Board
(CWQCB), San Francisco Regional Board, the following three samples were obtained:
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1) Effluent from a municipal wastewater treatment plant (Sample No.
AIHL-85-0403). The municipal wastewater was treated by chemical, physical,
or biological means prior to discharge. Three stages of treatments, primary,
secondary, and tertiary, were used at the municipal treatment plant. The
wastewater was characteristic of that from an urban community. Various
types of industrial discharges enter the system including those from some
small businesses and light industries such as painting, paper decorating, roofing,
sheet metal work, commercial printing, electroplating, metal finishing,
machinery, electronic, gasoline service stations, auto repair shops, disinfecting
and exterminating services, medical laboratories, etc. This sample may also
contain products from cooking greases, household chemicals such as cleaners,
detergents, auto/furniture polishes, drain openers, antifreeze, paint3 or thinners,
wood preservatives, pesticides, herbicides, pool chemicals, motor oil, radiator
flush, photographic chemicals, disinfectants, hair spray, lighter fluid, leather
conditioner, spot remover, windshield cleaner, solvents, etc., and human and
domestic wastes (Hathaway, 1980).
2) Effluent from an industrial wastewater treatment plant (Sample No. AIHL-85-
0406). Industrial wastewaters vary significantly in pollutant characteristics
according to the source of industry types, e.g., the industries in the
neighborhood of Berkeley and the San Francisco Bay Area include large varieties
of petroleum refineries, and chemical, pharmaceutical, agricultural, electronic,
and genetic engineering firms. Thus, chemical composition of each industrial
wastewater sample can be very different. The sample we evaluated was from
a plant mainly treating wastewater from a refinery. The petroleum industry
uses water for upgrading and refining crude oil. An average of 200 L of
water is required to refine each 42 gallon barrel of crude oil (Metcalfe et
al., 1985). The used water is then treated in an industrial wastewater treatment
plant. Therefore, the effluent sample probably contains residues from the
abovementioned processes (CDHS, 1983).
3) Surface runoff from a landfill (Sample No. AIHL-85-0405). This sample was
collected from a class I hazardous waste disposal facility. The landfill is a
disposal facility where hazarous waste is placed in or on the land; additionally,
the landfill does not have a land treatment facility, surface impoundment, or
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an injection well. By regulation, class I dump sites must provide complete
protection for the quality of ground and surface waters from all wastes
deposited therein and against hazard to public health and wildlife resources.
Wastes disposed of in a class I landfill contain toxic substances which could
significantly impair the quality of usable waters. Examples include but are
not limited to the following:
a) wastes of municipal origin, such as saline fluids from water or waste
treatment and reclamation processes, community incinerator ashes, and
toxic chemical toilet wastes;
b) wastes of industrial origin, such as brines from food processing, oil well
production, water treatment, industrial processes, and geothermal plants;
process ashes, chemical mixtures, mine tailings from which toxic
materials can leach; and rotary drilling muds;
c) wastes of agricultural origin, such as pesticides, fertilizers, and discarded
containers of chemicals;
d) other toxic wastes such as compounds of arsenic or mercury or chemical
warfare agents (Franks, 1981).
Two samples were collected by AIHL staff:
Aqueous fraction from a solid waste extraction procedure. A Toxicity
Characteristics Leachate Procedure (TCLP) was performed on a National Bureau
of Standards (NBS) reference sludge sample. The aqueous extract of the TCLP
procedure is used as a simulated landfill leachate wastewater sample (Sample
No. AIHL-85-0401). TCLP is one of the solid waste extraction procedures
developed under the Resource Conservation and Recovery Act (RCRA). The
NBS sludge sample was provided by Dr. Llewellyn R. Williams, USEPA, Las
Vegas, Nevada.
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The TCLP is similar to the Extraction Procedure (EP, 40 CFR 261.24, specified
in RCRA Section 3001) developed by the Office of Solid Wastes, EPA (USEPA,
SW-846, 1982). EP toxicity is one of the four characteristics established by
EPA for identifying hazardous waste (40 CFR 261.20). To emplement the EP
toxicity characteristic, EPA developed a mandatory testing procedure which
extracts the toxic constituents from a solid waste in a manner believed to
simulate the leaching action that occurs in landfills. The EP toxicity testing
protocol subjects a representative sample of a solid waste to an acidic leaching
medium.
Like the EP, the TCLP is designed to simulate the leaching a waste will
undergo if disposed of in a sanitary landfill. The following procedures (according
to a draft of TKO703, revised on 3/2/85) were used in this project, (a) The
liquid phase of the NBS sludge sample (16 bottles, labeled as: EPA/N3S
Reference Sludge, RCRA EP-Inorganics, September 1981) was separated by
centrifugation and filtration (MILLIPORE membrane filters, 0.45 pm pore size)
from the solid phase and was stored in a refrigerator for later analysis, (b)
The wet solid phase was weighed. If necessary, the particle size of the solids
can be reduced by crushing or by grinding, (c) The solids were mixed with
acetate buffer (0.1N, pH 5). Originally, the buffer was prepared according
to the following formula by EG&G PRINCETON APPLIED RESEARCH: Dissolve
8.2 g of anhydrous sodium acetate in 800 mL deionized water and adjust to
pH 5 with glacial acetic acid; then dilute to 1 L with deionized water. We
replaced the deionized water with ASTM Type I water which is a better grade.
(See Section 6.1.1.6, EPA/NBS Reference Sludge - TCLP Leachate, on
discussions of problems encountered by using this formula). The volume of
the buffer used equals 20 times the weight of the solids, (d) The extraction
was performed at room temperature using a shaker table for 18 hours, (e)
The sample was then centrifuged and filtered through a MILLIPORE membrane
filter (0.45 pm pore size) using a sinter glass filter holder (See Section 6.1.1.6
for details of problems), (f) The liquid phase was combined with the original
liquids from the sludge sample and served as the TCLP extract leachate.
Freshly prepared TCLP leachate was used for each liquid-liquid extraction
evaluation experiment.
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2) Brackish surface water receiving industrial effluents and areal runoff. Two
manual grab samples were collected as representatives of this type. One was
a San Francisco Bay surface water collected at the Berkeley Marina (Sample
No. AIHL-B5-0404). Another was collected at the discharge site in the San
Francisco Bay of an industrial wastewater treatment plant (Sample No.
AIHL-85-044A). A boat was used to get access to the site. Each sample
was collected from a pristine environment on a sunny autumn day in the
morning.
San Francisco Bay is located at the mouth of the Sacramento-San Joaquin
river system, a major estuary greatly modified by human activity. In 1978,
wastes from more than 30 municipal and 40 industrial waste treatment facilities
and from an additional 100 smaller industrial dischargers entered the bay at
nearly 4 percent of the average annual freshwater inflow. Untreated urban
runoff also enters the bay through more than 50 small local streams. Additional
contamination results from daily accidental spills of industrial chemicals and
oil (Nichols et al., 1986).
Two contaminated groundwater samples were obtained under the auspices of
the Toxic Substances Control Division, CDHS, and the Hazardous Materials Laboratory
(HML), CDHS. Two samples from the Stringfellow Quarry Hazardous Waste Disposal
Site near Riverside, California, were sent to AIHL by the on-site contractor, Science
Applications International Corporation (SAIC). One sample (Sample No. AIHL-85-0402)
was collected from an on-site (OW-2) well. Another (Sample No. AIHL-85-042A)
was from an upstream well (UGB-8).
The Stringfellow industrial waste disposal facility, situated in the Pyrite
Canyon, is located in Riverside County, approximately five miles northwest of the
city of Riverside. It is one of the most notorious uncontrolled hazardous waste sites
in the nation (Ember, 1985). During the operation period from August 21, 1956,
until voluntary closure in 1972, the site accepted about 34 million gallons of industrial,
agricultural, and Defense Department wastes. Companies with products such as
chemicals, solvents, pesticides, aircraft, steel, aluminum and other heavy metals,
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and insulation materials dumped mixtures such as acid wastes, pesticides, chromate
wastes, paint sludge and thinner, water softening brine, caustic soda, cyanides, starch
sump residuals, etc., into surface ponds (Hatayama et al., 1979). The groundwaters
in the neighborhood area were originally used for irrigation and for other domestic
and industrial purposes. Analysis of the groundwater from nearby wells prior to
establishment of the site indicated quality ranging from excellent to good for the
beneficial uses. However, groundwater was contaminated as the result of contact
with the wastes and the contaminated soils. The contaminated groundwater seeped
through the fractured bedrock, and escaped the site.
CDHS, with the concurrence of EPA, has contracted with SAIC of La Jolla,
California, to perform a remedial investigation/feasibility study on the site. SAIC
collected two samples for AIHL to be used in this protocol evaluation study. One
contaminated groundwater sample was collected from an on-site extraction well,
OW-2, in Pyrite Canyon. Another was collected from an upgradient extraction well,
UGB-8, outside the north boundary of the site. Previous chemical analysis results
indicate that OW-2 is one of the most contaminated wells and that UGB-8 contains
much lower concentrations of contaminants. The extraction wells are equipped with
submersible pumps and are operated routinely. In all cases, wells were purged prior
to sampling to ensure collection of representative samples. Sampling wells were
purged until constant readings were obtained for pH, for electrical conductivity, for
redox, and for temperature. After purging for approximately 20 gallons of discharge
water, the sample was collected from the sampling spigot on the well-head. After
collection, sample containers with appropriate labels were then inspected, were
chain-of-custody sealed, and were stored in ice chests for transport (Shokes, 1984).
The samples were shipped at ambient temperature to AIHL by Federal Express.
SAMPLE COLLECTION, LOG-IN, AND STORAGE
It is recommended that samples be taken which reflect the "normal" state of
the sample site. Periodic sampling of the same site over several months may also
be valuable in providing information on peak periods of activity (deVera et al., 1980).
For aqueous samples, the most common sampling procedure is a manual grab collection
of the volume needed for analysis. Amber glass bottles (1 gallon capacity) were
most useful for handling, shipping, storage and processing. These bottles were rinsed
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with ASTM Type I water, pesticide-grade methanol, hexane, and finally dlchloro-
methane before use. Bottles were air-dried and were capped using the original bottle
cap lined with a Teflon insert. All bottles were clearly labeled with the date and
time of sampling, the site, and the field manager's name and telephone number if
available. Once collected, samples were 9ealed in the amber glass bottles and were
held at 4°C during storage. The head space in the container was reduced by
completely filling the container with the sample or by replacement of air with a
nitrogen blanket. Table 2 lists the volume of sample required. At the time of
collection, a sample collection record form was filled out by the field manager
including any pertinent information relating to sample collection procedures, sample
type and appearance, any necessary treatments, and storage and shipment methods.
All of the completed forms obtained in this project are attached in Appendix C. A
copy of the form accompanied the sample through all phases of the processing and
the bioassay to insure the chain-of-custody.
Shipping and storage of the sample was planned 30 a3 to minimize the length
of time from collection to processing. Shipping and storage at 4°C is recommended,
and protection from light sources is mandatory. It was originally recommended that
all samples be processed (separated, extracted, and concentrated) within 14 days
after collection. However, our results suggest that mutagens in some samples may
decay during storage. Thus, it is recommended that the storage time should be
reduced to the minimum time possible, e.g., the sample should be extracted 24 hours
after receipt if possible. The 24-hour period is needed for gravity separation of
solid and liquid phases. It is also recommended that samples be completely analyzed
within 40 days of processing (See Section 6.1.1.7, Possible Changes in Sample
Composition, for discussions).
PHASE SEPARATION
Each sample container was stored in the dark, motionless at 4°C for 24 hours
after receipt. During storage, a gravity separation of phases occurred. All the
samples we collected so far do not contain any nonaqueous liquid phase and contain
either no or less than 5% suspended particles without solid sediments at the bottom
of the container. Therefore, the protocol evaluation effort has been concentrated
on the liquid-liquid extraction process.
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TABLE 2. RECOMMENDED VOLUMES AND STORAGE AND SAMPLES
Component
Volume or Weight
Storage Conditions
Collected Sample
Gravity Separation Method
Sample for XAD-Resin
Column Concentration
Liquid-Liquid Extraction
Method
Solids for Solid Waste
Extraction
Extracted/Concentrated/
Solvent Exchanged
Sample for Bioassay
Minimum of 30 L
Minimum of 30 L
10 L
3 L
500 g net weight
10 mL
4 C, dark
4°C, dark motionless
4°C, dark glass or
Teflon-lined vessel
4 C, dark glass or
Teflon-lined vessel
4°C, dark closed
container
4°C, amber glass vial
with Teflon cap liner
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The XAD method as described in the Drinking Water Protocol (Appendix B)
requires a 10 L sample and a set of expensive stainless steel columns. It is more
time-consuming to process when compared to the liquid-liquid extraction method.
In addition, the XAD resin requires extensive multi-steps of pre-cleaning. In principal,
the XAD method can concentrate larger samples; and it may provide greater
concentration than liquid-liquid extraction. However, as recommended in Section
3.1.1, XAD-Resin Method, further studies are needed to understand several problems
with the XAD method. On the other hand, the liquid-liquid extraction procedure
requires only a 3 L sample, is less expensive to set up, and takes less time to
perform. Because of these advantages, the liquid-liquid extraction method was
evaluated first with six generic types of wastewater samples.
Samples were processed based on the procedures indicated in the flow chart
on Figure 2. The sample was separated into several aliquots. One aliquot of the
sample was prepared according to the liquid-liquid extraction protocol. If the result
from the Ames assay was positive, another aliquot of the sample was extracted using
the identical procedures. The replicate, or triplicate when possible, results were
compared for precision expressed by SD. A sample with relatively stable mutagenic
activity was used as the base material for spike mutagen recovery studies. Field
blanks and reagent blanks were evaluated to establish the background of preparation
methods.
If the result of the first extraction and mutagen screening experiment was
negative or equivocal, modifications of the extraction or mutagenicity testing
procedures were made for the second experiment. If the results of the follow-up
experiments were negative, another aliquot could be evaluated using the XAD-resin
concentration protocol.
The liquid-liquid extraction methods for the processing of wastewater samples
for the application to the Ames assay are described below essentially as they appear
in the consensus protocol (Appendix A). Some minor revisions and modifications of
the liquid-liquid extraction procedure have been suggested and have been validated.
These changes are described in Section 6.1, Wastewater Protocol Evaluation, as part
of the protocol validation.
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OTHER
ALIQUOTS
AFTER THE FIRST
PROCESSING, IF
POSITIVE, REPEAT
SAME PROCEDURES;
F NEGATIVE, MODIFY
PROCEDURES
AMES
TEST
NEGATIVE AT THE
ECONO PROCESSING
^ FIRST AND ^
SECOND ALIQUOTS
SAMPLE COLLECTION
COLLECTED SAMPLE
LIQUID-LIQUID
EXTRACTION
XAD-RESIN
CONCENTRATION
SAMPLE LOG-IN AND STORAGE
POSrriVE AT THE
SECOND PROCESSING
OR NEGATIVE AT THE
THIRD PROCESSING
ANALYSIS AND REPORT
Figure 2. Sample processing scheme.
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LIQUID-LIQUID EXTRACTION
Processing of wastewater samples by liquid-liquid extraction (See Figure 3)
was carried out in a chemical fume hood and under yellow lights. Appropriate safety
precautions as listed on the EPA-consensus protocol were complied with carefully.
All processing was performed at room temperature. Two 15Q0 mL aliquots were
measured from the aqueous sample. The sample was transferred into two 2000 mL
glass beakers with each containing a 2" Teflon coated stir bar. The contents of
both beakers were equilibrated to room temperature (approximately 20°C to 25°C).
The initial pH of the sample was determined using a pH meter with stirring. The
pH of the water was adjusted to 11 with 5N NaOH. The aqueous phase was then
transferred to two 2000 mL separatory funnels for extraction.
One hundred and fifty mL of dichloromethane was added to each separatory
funnel after rinsing the beakers with the solvent. The basified sample was extracted
by shaking the funnels for two minutes with periodic venting to release pressure.
The organic layers were allowed to separate from the water phases for a minimum
of ten minutes. The dichloromethane extracts were combined in a 1000 mL Erlenmeyer
flask. The flask was kept in a hood. One hundred mL of dichloromethane was
added to each separatory funnel, and the extraction procedure was repeated a second
time. All extracts were combined in the 1000-mL Erlenmeyer flask. A third 100
mL extraction was performed in the same manner. The pH of the aqueous phase
was adjusted to less than 2 using a diluted (1:1) sulfuric acid solution. The acidified
sample was serially extracted with 150, 100, and 100 mL of dichloromethane per
separatory funnel. All the extracts were combined in the 1000-mL Erlenmeyer flask.
Notice that additional fractionation could be achieved at this stage by not combining
these extracts (organic acids) with previous extracts (organic base/neutrals) but,
rather, by processing each separately through the remaining procedure. Separate
processing of acid and base/neutral fractions may be required with some samples.
A Kudema-Danish (K-D) concentrator was assembled by attaching a 25-mL
concentrator tube to a 500-mL evaporative flask. Other concentration devices such
as a rotary evaporator may be used in place of the K-D (See Section 6.1.2.4, Replace
K-D Apparatus with Rotary Evaporator, for discussions). The combined extract was
poured through a drying column containing about 10 cm of anhydrous sodium sulfate
-24-
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SAMPLE (3 liter)
1) Measure pH.
2) Adjust to pH 11 with NaOH.
3) Extract with dichloromethane.
Aqueous Fraction
1) Adjust to pH 2
with H2S04
2) Extract with
dichloromethane
Organic Base/Neutrals
Aqueous Fraction
(Discard)
Organic Acids
1) Combine (or not combine,
if requested) fractions
Organics
1) Pass through an anhydrous
Na2S0^ column.
2) Concentrate with Kuderna-
Danish apparatuses.
3) Transfer to a glass vial.
4) Dried under nitrogen.
5) Weigh and dissolve
residue in DMSO.
v
Ames assay
Figure 3. Liquid-liquid extraction scheme.
-25-
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on top of 2 cm glass wool, and the extract was collected in the K-D concentrator.
One or two clean Teflon-coated boiling chips were added, and a three-ball Snyder
column was attached. The Snyder column was primed by adding about 1 mL of
dichloromethane to the top of the column. The K-D apparatus was placed on a hot
water bath (60°C to 70°C) so that the concentrator tube was partially immersed in
the hot water, and the entire lower rounded surface of the flask was bathed with
hot vapor. A 2-L beaker was a suitable water bath. In addition, the lower 500 mL
evaporator flask was covered with aluminum foil forming a tent over the beaker
water bath. In this way, the steam formed from the water bath will bathe the
flask more efficiently. At the proper rate of distillation, the balls of the column
will actively chatter, but the chambers will not flood with condensed solvent.
The Erlenmeyer flask and the sodium sulfate column were rinsed with 25 mL
of dichloromethane to complete the quantitative transfer. Concentration on the
water bath was continued until the liquid reached less than 10 mL. The K-D
apparatus was removed from the water bath and was allowed to drain and to cool
for at least 10 minutes. The Snyder column was removed, and the flask with its
lower joint into the concentrator tube was rinsed with 1 to 2 mL of dichloromethane.
The K-D apparatus was adequate for evaporation purposes. However, dichloromethane
has been reported as an animal carcinogen and should be U9ed with extra caution.
Alternatively, the rotary evaporator technique is recommended so the potential
carcinogenic solvent can be collected and can be disposed of properly.
The extract was transferred to a tared, 3-dram glass vial, and the remaining
dichloromethane was evaporated under nitrogen using a heating block or water bath
to prevent sample cooling. The temperature was not allowed to exceed 35°C. The
vial and its contents was weighed, and the weight of the residue was recorded. If
requested, the residue can be dissolved in 10 mL dichloromethane, and an aliquot
can be removed for total organic carbon analysis (American Public Health Association
et al., 1985), and an 1 mL aliquot can be analyzed by gas chromatography/mass
spectrometry using EPA Method 625 (44 CFR 233, December 3, 1979; Longbottom
and Lichtenberg, 1982).
The dried residue was stored in the dark at -20°C until Explication to the
Ames assay. If no aliquot was removed for analysis by gas chromatography or for
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total organic carbon analysis, resuspension of the residue in a 1.0 mL vehicle (e.g.,
DMSO for Ames assay) represents a concentration factor of 3000-fold.
AMES ASSAY
Assay Scheme
As shown in Figure 4, a strategic Ames assay scheme was developed (Zeiger
et al., 1985b) for optimum cost- and effort-effectivness. All fractions were tested
for mutagenic activity using Salmonella typhimurium strains TA98 and TA100 in the
standard Ames plate incorporation assay. Due to limited sample size, only these
two strains were selected for screening the complex wastewater samples. All samples
were initially assayed in the presence and the absence of an exogenous metabolic
activation system (2% and 10% S-9 fraction in the S-9 mix) derived from the livers
of Aroclor 1254-induced Sprague-Dawley rats (purchased from LITTON BIONETICS,
Kensington, Maryland). The protein concentration of the S-9 used in this project
was 25 mg/mL as determined by the method of Lowry et al. (1951). All samples
were evaluated by using a minimum of three dose levels at half-log intervals and
by using triplicate or duplicate plates per dose whenever possible. Doses as high
as 6 mg extract/plate were used when the sample quantity was sufficient.
If a positive response was seen in the initial screening test, only the strain
giving the greatest response was used in the repeat assay with the same level of
S-9 fraction. If a questionable mutagenic response was observed only in the presence
of metabolic activation (2% or 10% S-9 mix), the strain(s) producing this response
was retested using the same level of S-9 with higher nontoxic doses or with 30%
S-9 mix. The dose ranges selected for the repeat test were based on the results
obtained in the original test. If a questionable positive response was observed only
in the absence of the metabolic activation system, the strain(s) producing this response
was retested without S-9 by using an adjusted dose range. If a negative and toxic
response was seen with and without the metabolic activation systems, the test was
repeated by using 30% S-9 mix. If a negative and nontoxic response was seen at
lower doses, the test was repeated with increasing doses up to 6 mg extract/plate
both in the presence and in the absence of 10% S-9 mix.
-27-
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Initial Assav
Strains
TA98,
TAIOO
5-9 mix
without S-9
with 2% S-9
with 10% S-9
Doses (un/extract/plate)
1, 10, 33, 100, 333, 1000,
when sample amount is sufficient.
Duplicate or triplicate plates per
dose if possible.
Repeat Assay
positive
results
Repeat only the strain
and condition in which
the maximum activity
is observed
negative
results
Nontoxic at
high dose(s)
Increase the dose(s) up
to 6 mg/plate if possible
and test with the
optimum level
Toxic at
high dose(s)
Repeat with 30%
mix
Figure 4. Ames assay scheme.
-28-
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QC of the Ames Assay
The Salmonella typhimurium strains U9ed are all histidine auxotrophs by virtue
of mutations in the histidine operon. When these histidine-dependent cells are grown
on minimal medium agar plates containing a trace of histidine, only those cells that
revert to histidine independence (his"*") are able to form colonies. The small amount
of histidine allows all the plated bacteria to undergo a few divisions; and this growth
is essential for mutagenesis to occur. The his4 revertants are easily visible as
colonies against the slight background growth. The spontaneous mutation frequency
of each strain is relatively constant (McCann et al., 1975a), but when a mutagen is
added to the agar, the mutation frequency is increased usually in a dose-related
manner.
The tester strains were obtained from Dr. Bruce N. Ames of the University
of California at Berkeley (Ames et al., 1975). In addition to having mutations in
the histidine operon, all the indicator strains have a mutation (rfa) that leads to a
defective lipopolysaccharide coat; they also have a deletion that covers genes involved
in the synthesis of the 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, thereby increasing the mutagenic effect of these molecules. The
uvrB mutation renders the bacteria unable to use the accurate excision repair
mechanism.
Strains TA100 and TA98 were used in this project because of their high
sensitivities for mutagens in complex environmental mixtures. Strain TA100 is derived
from TA1535 by the introduction of the resistance transfer factor, plasmid pKMlOl.
TA1535, carrying the base-pair substitution mutation at the hisG46 locus, is reverted
to his* by many mutagens that cause base-pair substitutions. The plasmid pKMlOl
is believed to cause an increase in error-prone DNA repair that leads to many more
mutations for a given dose of most mutagens (McCann et al., 1975b; Mortelmans
and Stocker, 1979). In addition, the plasmid confers resistance to the antibiotic
ampicillin. This antibiotic-resistant capability is a convenient marker to detect the
presence of the plasmid in the cells. TA100 can detect mutagens such as benzyl
chloride and 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) that are not detected by
TA1535. The presence of this plasmid also makes strain TA1Q0 sensitive to some
-29-
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frameshift mutagens, e.g., ICR-191, benzo(a)pyrene, aflatoxin B^, and 7,12-dimethyl-
benz(a)anthracene. Strain TA98 is derived from strain TA1538 by the addition of
plasmid pKMlOl. TA1538 carries a frameshift mutation at the hisD3052 locus.
All indicator strains are kept frozen in broth supplemented with 10% sterile
glycerol or DMSO at -80°C in 1-mL aliquots containing about 10^ cells. New frozen
stock cultures are made approximately every six months from single colony isolates
that have been checked for their genotypic characteristics (his, rfa, uvrB, bio) and
for the presence of the plasmid.
Some variation has been found in the extent of growth of bacterial strains in
overnight nutrient broth cultures. This seems to result from variability in the
nutritional quality of the medium. There seem to be marked differences not only
between media from different sources but also among batches from a single source.
Overnight cultures which have just reached a density of (1-2) x 10 viable cells per
mL are considered most desirable for mutagen testing.
For each experiment, the 1-mL frozen aliquots of the strain were allowed to
thaw at room temperature before inoculation into 50 mL of Oxoid nutrient broth
liquid medium. The cultures were grown at 37°C, were unshaken for up to 4 hours,
and then were gently shaken (100-120 rpm) for approximately 12 hours. Inocula
were taken directly from nutrient broth cultures. A fresh cell suspension was used
for each day's experiments. Cell suspensions were maintained through the day at
ice-bath temperature since storage at room temperature may result in loss of viability
or mutagen sensitivity or both (CDHS, 1979; CDHS, 1985). For QC purposes, cell
titer and viability was measured for each experiment (See Appendix F for data).
To ensure the validity of the test, the strain genotype function tests were
performed for each experiment. The procedures outlined in the methods paper (Ames
et al., 1975) are satisfactory with regard to confirming histidine requirement, deep
rough character, and ultraviolet sensitivity of tester strains. All strains were
genetically analyzed whenever experiments were performed (See Appendix F for
data). Tests for the presence of the R factor conferring ampicillin resistance in
strains TA100 and TA98 were conducted conveniently by using commercially available
-30-
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filter paper disks containing 10 yg ampicillin (Zeiger et al., 1981). Ampiciliin-
contalning disks were placed In the center of petrl dishes which were overlaid with
each of the tester strains. Zones of inhibition should be observed with strains TA98
and TA100. As this procedure will not determine what fraction of the culture has
lost the R factor, it is important to check stock cultures periodically to ensure that
close to 100% of the bacteria contain the R factor. This can be done by replica
plating (preferably with 100 or more colonies) onto ampicillin-free plates and plates
containing 25 yg ampicillin per mL of medium (CDHS, 1985).
Triplicate plates of the following compound were tested as positive controls:
2-nitrofluorene (2NF) for TA98 without S-9, 2-aminofluorene for TA98 with 2%, 10%
and 30% S-9 mix, sodium azide for TA100 without S-9, and 2AA for TA100 with
2% or 10% S-9 mix and for TA98 with 30% S-9 mix. The data are recorded on the
result form in Appendix D and also are listed on the cell function test form in
Appendix F. Four conditions of S-9 were used to cover various requirements for
optimum enzyme activation of different chemicals in the complex mixture wastewater
samples (Sugimura and Nagao, 1980).
Several negative controls including the spontaneous mutation control and the
solvent control were performed for each experiment. The extract of the laboratory
distilled water which is used as the field, travel, and sample preparation method
blank was also tested. Laboratory QC charts were established for these controls
for accuracy and precision measurements. Statistical analyses were performed to
establish the method background (See Section 6.2.3, Statistical QC Analysis and
Records, for details).
As with the extraction procedures, mutagenicity testing was carried out in a
room fitted with yellow fluorescent lights to minimize potential photooxidation. All
the residues and chemicals were processed in an approved chemical fume hood.
Safety rules described in the EPA-consensus protocol were complied with carefully.
EXTRACTION EFFICIENCY
Extraction efficiencies for the recovery of mutagens from an environmental
water sample by the liquid-liquid extraction protocol can be assessed by two methods:
-31-
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(1) by chemical analysis such as high pressure liquid chromatography (HPLC) for
identification and quantitation of chemical standards; (2) by the Ames assay for the
recovery of mutagenicity. Both methods quantitate the level of the spiked mutagens
recovered in the final extract residue.
Chemical Analysis by HPLC
Chemical SPRM standards were provided by Dr. Llewellyn R. Williams, EPA.
They were separated, identified, and quantitated by reversed-phase HPLC using a
Varian model 5000 liquid chromatography The method used requires three reservoirs,
and, therefore, only liquid chromatography with this capability can be used. Five
chemical mutagens with diverse chemical properties have been separated. They were
4-nitroquinoline-N-oxide (4NQO), 2NF, B(a)P, 2AA, and 4NBA. However, only three
mutagens, B(a)P, 2AA, and 4NBA, were used in the recovery studies (See Section
6.1.3, Mutagen Extraction Efficiency, for details) because of time and resource
limitations. The recovery of other chemical mutagens will be evaluated in the
future. This HPLC scheme provides a basic method for the separation of numerous
chemical standards. The method reported is a general protocol to adequately separate
and quantitate the recovery standards reported in the results section.
All five of these compounds can be separated on an Altex ultrasphere-ODS-C18
column or its equivalent. Peaks were identified and were quantitated with a Varian
UV-50 multiple wavelength spectrophotometer and a Varian Fluorichrom fluorimeter
with a deuterium lamp supply. Filters were set for an excitation band of 340-380
nm and an emission cutoff of 460 nm for B(a)P fluorescense detection. The gradient
used is shown in Table 3, and the flow rate is 1.5 mL/min. A sample chromatogram
is shown in Figure 5.
Mutagenicity Recovery Analysis
Three SPRM's were used in the mutagenicity recovery studies: B(a)P, a neutral
chemical; 2AA, a basic chemical; and 4NBA, an acidic chemical. These chemicals
were chosen because of the fact that the liquid-liquid extraction is a method for
selectively extracting acidic, neutral, and basic compounds.
-32-
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TABLE 3. HPLC GRADIENT CONDITIONS FOR THE SEPARATION OF SPIKED
REFERENCE MUTAGENS
Step Time % Reservoir
(min) A B C
1 0 (Initial) 100% - 0% Injection
2 5 100% - 0%
3 50 0% 100%
4 60 - 0% 100%
5 65 - 100% 0%
6 75 - 100% 0%
7 77 - 90% 10%
8 78* 100% - 0% End
Stepwise gradient is dependent on a three-reservoir, paired system in which reservoir
A is 10% acetonitrile in 0.02M (NH^HPO^ (ammonium carbonate can be substituted),
reservoir 9 is distilled water, and reservoir C is acetonitrile. All solvents and buffers
are HPLC grade and should be filtered before use. Reservoir A should be degassed
by continuously purging with helium during the run.
•Run until equilibrated before subsequent injection.
-33-
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1 0
0 9
0 8
0 7
HPLC OF RECOVERY STANDARDS
INJECTED 20ng EACH
ABSORBANCE- 254nm
z
3 0 6
>•
X
<
a.
oo
OS o 5
<
ui
u
z
<
S °-4
o
(/)
a
<
0.3
0 2
<
u
5
N
z
Ui
a
O
(O
I
<
o
N
1
Z
UJ
o
40
TIME (MINUTES)
Figure 5. HPLC chromatograph of spiked reference mutagens.
-34-
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Prior to extraction, a 1.5 L aliquot of water was spiked with a known quantity
(0.25 ug-5 mg range) of one of the three chemical standards. The water was then
processed essentially by following the procedures described in Section 5.4, Liquid-
Liquid Extraction, and the modifications, when applicable, recommended in Section
6.1.2, Liquid-Liquid Extraction Results and Discussions. The doses (mg/plate) used
in the Ames assay of the recovery study (See Appendix E for primary data) were
estimated mg of the SPRM in the extract. The estimated doses were calculated
from the original spiked dose and were based on the assumption of 100% recovery
of the SPRM.
The percentage of recovery of the spiked mutagen was calculated by HPLC
peak analysis of both a known standard and the unknown residue. When sufficient
material was available, the mutagenicity recovery was also calculated from a
dose-response curve of the pure chemical standard under the same conditions as the
residue in the Ames assay. The conditions used in these studies for B(a)P and 2AA
were TA98 with 2% and 10% S-9 mix, and for 4NBA, TA100 without S-9.
SECTION 6
RESULTS AND DISCUSSIONS
WASTEWATER PROTOCOL EVALUATION
Wastewater samples were collected with the extracts prepared following the
procedures in the EPA proposed sample preparation protocol, and the extracts were
tested in the Ames/Salmonella assay. Each sample was accompanied by a sample
collection record that documented the sampling procedures (See Appendix C). For
all wastewater samples, the EPA-suggested preparation protocol was carried out at
least once, and additional procedures were developed to accommodate individual
samples as needed. These modifications are described below.
Ames Assay Results and Interpretation
As listed on Table 1 in Section 2, Conclusions, mutagenic activity is detected
in all the wastewater samples using the liquid-liquid extraction method. The primary
-35-
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data are attached as part of Appendix D. All the samples are more mutagenic in
bacterial strain TA98 than in TA100. The optimum S-9 condition for each sample
is also listed on Table 1. The mutagenic activity expressed as revertants per liter
of wastewater sample is obtained as follows: First the ASTM Type I blank water
is used to establish the method background. It serves as the field and the travel
blank as well as the method blank. Since the extract of the water is dissolved in
DMSQ, the water blank also includes the solvent (DMSO) blank (See Section 6.2.3,
Statistical QC Analyses and Records, for statistical evaluation and Appendix G for
primary data). A moderately conservative detection limit of two-fold of the method
background is then established (Ames et al., 1975; Dunkel and Chu, 1980; Margolin,
1985; Williams and Preston, 1983). Responses below the two-fold detection limit
are not considered for further evaluation. Positive responses are strengthened by
dose-response relationships (deSerres and Shelby, 1979). This is established by plotting
plate counts (y-axis, in revertants/plate) as a function of extract residue weight
(x-axis, in mg/plate). The method background in revertants/plate is used as the
response of the zero dose. The dose-response curves for all samples are also graphed
and are attached as part of Appendix D. The slope (b, as shown in the linear
regression calculation on the graphs in units of revertants/mg) of the linear portion
of the curve is used to estimate the optimum specific activity of the sample. Only
when the regression coefficient (r, as shown the the graphs) is > 0.9, the maximum
initial slope is considered acceptable. The residue weight (mg/L of original water
sample) is obtained and is recorded in the laboratory notebook. The final mutagenic
activity (revertants/L) is calculated by multiplying the slope (revertants/mg) times
the residue weight per liter.
The detection limit for the specific activity in units of revertants/mg is
calculated using net revertants/plate: Two times the background, corrected for the
response of the zero dose, is then divided by the maximum nontoxic dose tested.
The method background, established as described in Section 6.2.3, Statistical QC
Analysis and Records, was the water blank value. One exception was the TCLP
leachate. For that sample, the sodium acetate buffer blank value was used as the
zero value for the dose-response curve.
Sample characteristics observed upon receipt and several parameters measured
during sample preparation processing were tabulated for comparison (Table 4). The
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TABLE 4. WASTEWATER SAMPLE DESCRIPTION AND CHARACTERIZATION
WASTEWATER SAMPLE TYPES
Characteristics Industrial Municipal Contaminated Landfill Estaurine TCLP
Effluent Effluent Groundwater Runoff Brackish Leachate
pH Range
Color
Odor
Emulsion^
Precipitation
during ^
Processing
Residue
Weight (mg/3 L) 48-158
(Range)
7.1-7.5 6.2-6.5 3.2-3.5
Lt. Yellow Lt. Yellow Lt. Brown
Petroleum Sewage Sulfur
Light Heavy Light
9-17
+++
40-129
7.2-7.6 7.7-8.0 adjust to pH 5.0
Lt. Yellow None None
None Briny None
Moderate Moderate Moderate
++
11-31
2-7
5-6
1. Relative degree of emulsion formed between the aqueous and the organic phases
during processing. The pH at which the emulsion occurred varied with each
sample.
2. Relative severity of precipitation for each sample as the pH was adjusted to
11. Samples labeled as negative (-) did not precipitate as the pH was raised.
-------
sample collection records contain more detailed information on the collection,
appearance, and storage of the sample (See Appendix C).
Surface Runoff from a Class I Landfill—
As summarized in Table 5, the surface runoff sample was extracted according
to the original protocol (base extraction first). It was active in TA98 but not in
TA100. The optimum condition was without S-9. The presence of 2% or 10% S-9
mix decreased the mutagenic activity by approximately 25%. There was no toxicity
in the initial screening experiment at doses up to 0.666 mg/plate. Some elevated
plate counts were observed in TA100 in the absence and presence of 2% S-9 mix
but were not above the detection limit. However, modification of the extraction
scheme because of the precipitation problem (See Section 6.1.2.2, Samples Form
Precipitates, for details) in two later experiments resulted in a response below two
fold of the background. Toxicity was observed in the last experiment which was
performed approximately one month after receipt of the sample. The toxicity and
loss of activity may be due to changes in sample composition (See Section 6.1.1.7,
Possible Changes in Sample Composition, for discussions) which can be caused by
factors such as microbial degradation of mutagens, microbial formation of antagonists
(Alexander, 1974), chemical interference or chemical degradation, or combination of
these factors.
Brackish Surface Water Receiving Industrial Effluents—
The brackish San Francisco Bay surface water is an example of samples
requiring 2% S-9 mix for maximum activity. In the initial screening experiment,
the results of all six conditions tested were below the detection limit. However,
dose-related elevated colony counts were exhibited. Especially in the case of TA98
with 2% S-9 mix, the result (90 revertants/plate) was almost equal to the detection
limit (93 revertants/plate). This condition was therefore used in the follow-up
experiments. As shown on Table 6, a comparison of either acid first or base first
extraction method produced similar results. The San Francisco Bay receives local
industrial (mainly refinery) effluents which may be a major source of pollutants. A
brackish surface water sample (No. AIHL-85-0404) at the discharge site of an industrial
wastewater treatment plant was collected (No. AIHL-85-044A) and was tested. Both
acid first and base first procedures produced results similar to those of the previous
Bay water sample (Table 7).
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TABLE 5. MUTAGENICITY OF A SURFACE RUNOFF FROM A CLASS I LANDFILL, SAMPLE
NO. AIH_-85-0405
A. Results of the Initial Screening Experiment
2 3
Extraction Residue Sample Specific Mutagenic Activity (revertants/mg) '
Exp. Date Method* Weight Volume TA9B TA100
(mg) (L) -S9 2%S9 10%S9 -S9 2%S9 10%S9
9/24/85
Base/Acid
15
122
88
86
< 242 < 230 < 200
B. Comparison of Mutagenic Response in TA98 without S-9
Exp. Date
Extraction
Method^
Residue
Weight
(mg)
Sample
Volume
(L)
Specific Mutagenic Mutagenic Response
Activity . j
(revertants/mg) '
per Unit Volume
(revertants/L)
9/24/85
10/4/85
10/17/85
Base/Acid
Acid/Base
Acid/Base
15
31
11
122
< 11
< 32'
614
< 112
< 114
The extraction method indicates the order of pH in liquid-liquid extraction.
1.
2.
The specific activity of the mutagenicity dose response curve is represented by
the slope (b as shown in the statistical analysis on graphs in Appendix D).
3. The spontaneous mutation and the positive control values are listed on the Strain
Function Tests, Cell Titer, and Viability Record in Appendix F.
4. The optimum testing condition in the initial screening experiment was in TA98
without S-9 mix.
5. No revertant colonies on all the testing plates, background lawn was normal
except the plates at the highest dose.
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TABLE 6. MUTAGENICITY OF A BRACKISH SAN FRANCISCO BAY SURFACE WATER,
SAMPLE NO. AIHL-85-0404
A. Results of the Initial Screening Experiment
2 3
Extraction Residue Sample Specific Mutagenic Activity (revertants/mg) '
Exp. Date Method* Weight Volume TA98 TA100
(mg) (L) -S9 2%S9 10%S9 -S9 2%S9 10%S9
9/24/85 Base/Acid 6.5 3 < 106 < 139 < 162 < 484 <460 < 401
B. Comparison of Extraction Methods for Mutagenic Response in TA98 with 2% 5-9 Mix'*
Extraction Residue Sample Specific Mutagenic Mutagenic Response
Exp. Date Method* Weight Volume Activity ^ j Per Unit Volume
(mg) (L) (revertants/mg) ' (revertants/L)
10/4/85
Acid/Base
4.8
3
177
283
12/17/85
Base/Acid
1.2
1.5
218
176
1. The extraction method indicates the order of pH in liquid-liquid extraction.
2. The specific activity of the mutagenicity dose response curve is represented by
the slope (b as shown in the statistical analysis on graphs in Appendix D).
3. The spontaneous mutation and positive control values are listed on the Strain
Function Test3, Cell Titer, and Viability Record in Appendix F.
4. Although results of all six conditions in the initial screening experiment were
below the detection limit, TA98 with 2% S-9 mix produced a questionable positive
response close to the detection limit. This condition was therefore used in the
follow-up experiments.
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TABLE 7. MUTAGENICITY OF A BRACKISH RECEIVING SURFACE WASTEWATER FROM
THE DISCHARGE SITE OF AN INDUSTRIAL WASTEWATER TREATMENT PLANT,
SAMPLE NO. AIHL-85-044A
Comparison of Extraction Methods for Mutagenic Response in TA98 with 2% S-9 Mix^
Extraction Residue Sample Specific Mutagenic Mutagenic Response
Exp. Date Method^" Weight Volume Activity per Unit Volume
(mg) (L) (revertants/mg) ' (revertants/L)
11/20/85
Base/Acid
1.5
3
465
232
12/17/85
Acid/Base
0.9
1.5
414
240
1. The extraction method indicates the order of pH in liquid-liquid extraction.
2. The specific activity of the mutagenicity dose response curve is represented by
the slope (b as shown in the statistical analysis on graphs in Appendix D).
3. The spontaneous mutation and positive control values are listed on the Strain
Function Tests, Cell Titer, and Viability Record in Appendix F.
4. The optimum testing condition for the San Francisco Bay water was in TA98
with 2% S-9 mix. The same condition was applied to the brackish receiving
wastewater for comparison purposes.
-------
Municipal Wastewater Treatment Plant Effluent—
As summarized in Table 8, the municipal effluent wastewater sample was
mutagenic only in TA98 with 10% S-9 mix in the initial screening experiment. There
was no activity in TA100. No toxicity was observed at doses up to 1 mg/plate.
Similar results were obtained in two follow-up experiments. No toxicity was exhibited
at 2 mg/plate. Even though there was no precipitation at pH 11, the acid first
extraction scheme (See Section 6.1.2.2, Samples Form Precipitates, for details) was
compared with the original base first extraction because of the potential of the
municipal sample for stability in mutagen compositions. Both methods gave similar
mutagenic responses.
Industrial Wastewater Treatment Plant Effluent-
Several problems were encountered in the liquid-liquid extraction where the
EPA-suggested protocol was used without modification (See Section 6.1.2, Liquid-
Liquid Extraction, for discussions). Interestingly, the only sample residue that was
equivocal in the initial experiments without and with 2% and 10% S-9 mix (Table
9) was the only sample that did not exhibit any technical problems when using the
original protocol. This sample, an industrial wastewater treatment effluent, was
processed at first within three days of receipt. The sample had a strong petroleum-like
odor (Table 4). Elevated colony counts were observed but mutagenicity was not
significantly greater than twice that of background levels. The final residue
(approximately 50 mg/3 L) was dark red in color and possessed a strong mildew odor.
Toxicity was observed at the dose of 2 mg/plate. TA98 with 10% S-9 mix produced
response close to the detection limit and was considered as the best among the six
conditions. The residues obtained from the industrial wastewater sample within seven
days of receipt dissolved readily in DMSO. However, when this sample was processed
either by base first or acid first extraction again 24 days after receipt, the amount
of residue was significantly increased (approximately 120-160 mg/3 L). Furthermore,
the residue would not completely dissolve and this made analysis by the Ames assay
extremely difficult. After sonication, a fine particulate suspension of the residue
was applied for the Ames testing. However, there were no visible precipitates on
the plates after incubation. The results were again equivocal. Two more experiments
were attempted approximately four months after receipt. Because of light
precipitation and emulsion formation (See Section 6.1.2, Liquid-Liquid Extraction, for
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TABLE 8. MUTAGENICITY OF A MUNICIPAL WASTEWATER TREATMENT PLANT
EFFLUENT, SAMPLE NO. AIHL-85-0403
A. Results of the Initial Screening Experiment
Exp. Date Method
2 3
Extraction Residue Sample Specific Mutagenic Activity (revertants/mg) '
* Weight Volume TA98
(mg) (L) -S9 2%S9 10%S9
TA100
-S9
2%S9 10%S9
8/2/85 Base/Acid
17
<33 <43
88
< 166 <165 < 145
B. Comparison of Extraction Methods for Mutagenic Response in TA98 with 10% S-9 Mix
Extraction Residue Sample Specific Mutagenic
Exp. Date Method* Weight Volume Activity _ ..
(mg) (L) (revertants/mg) '
Mutagenic Response
per Unit Volume
(revertants/L)
8/2/85
8/13/85
10/17/85
Base/Acid
Base/Acid
Acid/Base
17 3
13 3
9 3
88
75
148
491
325
443
1. The extraction method indicates the order of pH in liquid-liquid extraction.
2. The specific activity of the mutagenicity dose response curve is represented by
the slope (b as shown in the statistical analysis on graphs in Appendix D).
3. The spontaneous mutation and positive control values are listed on the Strain
Function Tests, Cell Titer, and Viability Record in Appendix F.
4. The optimum testing condition in the initial screening experiment was in TA98
with 10% S-9 mix.
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TABLE 9. MUTAGENICITY OF AN INDUSTRIAL WASTEWATER TREATMENT PLANT
EFFLUENT, SAMPLE NO. AIHL-85-0406
A. Results of the Initial Screening Experiment
Extraction
Residue
Sample
2 3
Specific Mutagenic Activity (revertants/mg) '
Exp. Date
Method*
Weight
Volume
TA98
TA100
(mg)
(L)
S9 2%S9 10%S9
•S9 2%S9 10.%S9
9/24/85
Acid/Base
48
3 <
35 <46 < 54
< 161 < 153 < 134
4
B. Results of Follow-up Experiments in TA98 with 10% S-9 Mix
Extraction
Residue
Sample
Specific Mutagenic
Exp. Date
Method*
Weight
(mg)
Volume
(L)
Activity _ j
(revertants/mg) '
10/4/85
Base/Acid
52
3
< 53
10/17/85
Base/Acid
62
1.5
< 38
10/17/85
Acid/Base
79
1.5
< 38
C. Results
of Follow-up Experiments in TA98
with 30% S-9 Mix5
Extraction
Residue
Sample
Specific Mutagenic
Mutagenic Response
Exp. Date
Method*
Weight
(mg)
Volume
(L)
Activity - 3
(revertants/mg) '
per Unit Volume
(revertants/L)
2/11/86
Acid/Base
28
1.1
63
1585
2/28/86
Acid/Base
57
3
95
1810
1. The extraction method indicates the order of pH in liquid-liquid extraction.
2. The specific activity of the mutagenicity dose-response curve is represented by
the slope (b as shown in the statistical analysis on graphs in Appendix D).
3. The spontaneous mutation and positive control values are listed on the Strain
Function Tests, Cell Titer.and Viability Record in Appendix F.
4. Although results of all six conditions in the initial screening experiment were
below the detection limit, TA93 with lO^o S-9 mix was the best condition in
which the result was closest to the detection limit. This condition wa3 therefore
used in the follow-up experiments.
5. Modification of the S-9 condition (30% S-9 mix) was applied because of equivocal
results obtained in experiments with 10"'o S-9 mix.
-44-
-------
discussions), acid first extraction was applied. There were no solubility problems,
and the residue weights were 76 and 57 mg/3 L, respectively. Increasing the S-9
concentration to 30% was a necessary modification for detecting the mutagenicity.
Petroleum distillate extracts have been reported to be exhibiting significant mutagenic
response only at S-9 concentrations over 20% (Carver et al., 1985). Our results may
indicate the possible existence of similar chemicals in the industrial wastewater
treatment plant effluent sample.
Contaminated Groundwater from the Stringfellow Hazardous Waste Disposal Facility—
The Stringfellow on-site contaminated groundwater sample (No. AIHL-85-0402)
produced approximately 3600 revertants/L. Table 10 summarizes the results of the
four experiments performed on this sample. The residue contains both direct- and
indirect-acting mutagens. Dose-related elevated colony counts were observed in
TA100, but the numbers were less than twice the background. TA98 with 2% S-9
mix was the optimum Ames testing condition. Toxicity was not observed at doses
up to 3 mg/plate in the initial screening experiment.
The sample was fairly acidic (approximately pH 3.5). It precipitated heavily
at pH 11 which caused extreme difficulty in the basic extraction process (See Section
6.1.2.2, Samples Form Precipitates, for discussions). A modification in which the
acid extraction was applied first allowed easier separation between the organic and
aqueous phases. Three experiments using the modified extraction procedures were
performed. In the first follow-up experiment, most of the mutagenic activity was
found in the acid with neutral (pH 2) fraction. However, a large portion of the
mutagenicity was lost in comparison with the initial screening experiment in TA98
with 2% S-9 mix. Mutagenicity was also found in TA100.
In addition to the sample precipitation problem, this sample may have undergone
changes in chemical composition during storage (Table 10B, see Section 6.1.1.7,
Possible Changes in Sample Composition, for discussions). The mutagenic activity
gradually decreased over time to below the detection limit, and toxicity was observed
at doses equal and higher than 3 mg/plate.
A chemical analysis by gas chromatography by J. Tang and H. Okamoto at
HML, CDHS, according to EPA method 625 was performed on the Stringfellow
-45-
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TABLE 10. MUTAGENICITY OF A CONTAMINATED GROUNDWATER FROM THE STRING-
FELLOW HAZARDOUS WASTE DISPOSAL FACILITY ON-SITE WELL OW-2,
SAMPLE NO. AIHL-85-0402
A. Results of the Initial Screening Experiment
2 3
Extraction Residue Sample Specific Mutagenic Activity (revertants/mg) '
Exp. Tate Method* Weight Volume TA98 TA100
(mg) (L) -S9 2%S9 10%S9 -S9 2%S9 10%S9
7/22/85 9ase/Acid 129 3 58 84 79 < 57 < 61 < 56
4
B. Comparison of Mutagenic Response in TA98 with 2% 5-9 Mix
Extraction Residue Sample Specific Mutagenic Mutagenic Response
Exp. Date Method* Weight Volume Activity ^ 3 Per Unit Volume
(mg) (L) (revertants/mg) ' (revertants/L)
7/22/85 Base/Acid 129 3 84 3598
8/2/85 Acid/Base 43 3 48 685
pH 2 fraction only
8/2/85 Acid/Base 7 3 <41 <95
pH 11 fraction only
9/24/85 Acid/Base 60 4.5 <53 < 702
10/17/85 Acid/Base 67 3 <28 < 629
1. The extraction method indicates the order of pH in liquid-liquid extraction.
2. The specific activity of the mutagenicity dose-response curve is represented by
the slope (b as shown in the statistical analysis on graphs in Appendix D).
3. The spontaneous mutation and positive control values are listed on the Strain
Function Tests, Cell Titer, and Viability Record in Appendix F.
4. The optimum testing condition in the initial screening experiment was in TA98
with 2% S-9 mix.
-46-
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contaminated wastewater sample (No. AIHL-85-0402) and was confirmed by gas
chromatography/mass spectrometry-mass selective detector by J. Hennings and T. Li
at HML (CDHS, 1984). The following priority pollutants were searched and none
was detected in the blank water extract control: bis(2-chloroethyl)ether, 1,3-
dichlorobenzene, 1,4-dichlorobenzene, 1,2-dichlorobenzene, bis(2-chloroisopropyl)ether,
hexachloroethane, N-nitrosodi-n-propylamine, nitrobenzene, isophrone, bis(2-chloro-
ethoxy)methane, 1,2,4-trichlorobenzene, naphthalene, 2-chloronaphthalene,
acenaphthylene, dimethylphthalate, 2,6-dinitrotoluene, acenaphthene, 2,4-dinitro-
tolune, fluorene, hexachlorobutadiene, 4-chlorophenylphenylether, 4-bromophenyl-
phenylether, hexachlorocyclopentadiene, hexachlorobenzene, phenanthrene,
anthracene, di-n-butylphthalate, fluoranthene, benzidine, pyrene, butylbenzylphthalate,
1,2-benzanthracene, 3,3'-dichlorobenzidine, chrysene, bis(2-ethylhexyl)phthalate, di-n-
octylphthalate, benzo(a)pyrene, indeno(l,2,3-c,d^)yrene, l,2:5,6-dibenzoanthracene,
1,12-benzoperylene, phenol, o-chlorophenol, o-cresol, p-cresol, 2-chloro-5-methyl-
phenol, o-nitrophenol, 2,4-dimethylphenol, 4-ethylphenol, 2,4-dichlorophenol, 2,5-
dichlorophenol, 3-chlorophenol, 2,6-dichlorophenol, 4-chloro-2-methylphenol,
4-chloro-3-methylphenol, 2,3,5-trichlorophenol, 2,4,6-trichlorophenol, 2,4,5-trichloro-
phenol, 2,3,4-trichlorophenol, 3,5-dichlorophenol, 2,3,6-trichlorophenol, 3,4-dichloro-
phenol, 2,4-dinitrophenol, 4-nitrophenol, 2,3,5,6-tetrachlorophenol, 2,3,4,5-tetrachloro-
phenol, 3,4,5-trichlorophenol, pentachlorophenol.
Only trace amounts of the following four among the abovementioned chemicals
were identified in the Stringfellow sample: 1,2-dichlorobenzene, 1,4-dichlorobenzene,
isophorone, and 3-chlorophenol. Haworth et al. (1983) reported 1,2-dichlorobenzene,
1,4-dichlorobenzene, and 3-chlorophenol as nonmutagens for Salmonella typhimurium.
No mutagenicity test data was found for isophorone in a literature search by the
Environmental Mutagen, Carcinogen, and Teratogen Information Program (EMIC), Qak
Ridge, Tennessee. However, as an unsaturated ketone, isophorone is expected to
have a strong tendency to behave as a direct alkylating agent. This possible alkylating
activity suggests that this chemical may have mutagenic potential (Federal Register,
1979). Further investigation is needed to elucidate the mutagen identities in the
Stringfellow sample.
Several metal scans which analyze 16 potentially toxic metals using induced
coupled plasma atomic emission spectroscopy (USEPA, 1982) by M. Iskander at HML
-47-
-------
(CDHS, 1984) indicated high levels of chromium, zinc, nickel, and copper in the
original contaminated groundwater sample (No. AIHL-85-0402). However, the
dichloromethane extract which was applied in the Ames assay did not contain metals
at levels significantly higher than the distilled water blank control extract. Therefore,
the mutagenic activity in the Stringfellow sample was not caused by metal salts
(e.g. CrO^~).
The cause for the decrease of mutagenicity in the Stringfellow on-site well
contaminated groundwater sample may be any combination of chemical reactions,
microbial activities, or storage conditions during shipping and storage. A time-course
study is needed to confirm the degree of the degradation problem and to evaluate
the optimum storage condition and the maximum storage time. Before establishing
these optimum conditions, it is recommended that the sample processing be started
24 hours after receipt. The 24-hour wait is needed for gravity separation as suggested
in the original protocol.
The second Stringfellow groundwater sample (No. AIHL-85-042A) collected
from an upgradient well exhibited much lower mutagenicity (Table 11) when it was
compared to the on-site sample. No toxicity was exhibited at doses ud to 0.666
mg/plate. This sample may serve as a background level of mutagenicity measurement
for the Stringfellow site. Mutagenicity comparison of samples collected from different
spots in a hazardous waste site may provide valuable information for potential health
effect evaluation and for assisting in monitoring the spread of toxic contaminants.
EPA/NBS Reference Sludge - TCLP Leachate—
The TCLP leachate sample in the initial experiment exhibited high mutagenic
acitivity in both TA98 and TA100 (Table 12). Both direct- and indirect-acting mutagens
were detected. No toxicity was observed at doses up to 0.333 mg/plate. The
optimum testing condition was in TA98 with 2% S-9 mix. The addition of 2% S-9
mix increased the direct-acting mutagenicity by approximately 45%. However, the
extract of the sodium acetate buffer which was the medium for the TCLP extraction
produced colony counts two to five fold of the water blank values. The activity
was especially obvious in TA98 without S-9. An investigation tracing back through
the source of the error was performed so that corrective actions could be taken
before the data was accepted.
-48-
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TABLE 11. MUTAGENICITY OF A CONTAMINATED GROUNDWATER FROM THE STRING-
FELLOW HAZARDOUS WASTE DISPOSAL FACILITY UPGRADIENT WELL UGB-8,
SAMPLE NO. AIHL-85-042A
l>
Mutagenic Response in TA98 with 2% S-9 Mix
Extraction Residue Sample Specific Mutagenic Mutagenic Response
Exp. Date Method* Weight Volume Activity ^ 3 Per Unit Volume
(mg) (L) (revertants/mg) ' (revertants/L)
10/17/85 Acid/Base 3.8 3 138 174
1. The extraction method indicates the order of pH in liquid-liquid extraction.
2. The specific activity of the mutagenicity dose-response curve is represented by
the slope (b as shown in the statistical analysis on graphs in Appendix D).
3. The spontaneous mutation and positive control values are listed on the Strain
Function Tests, Cell Titer and Viability Record in Appendix F.
4. The optimum testing condition for the Stringfellow OW-2 sample was in TA98
with 2% S-9 mix. The same condition was applied to the UGB-8 sample for
comparison purposes.
-------
TABLE 12. MUTAGENICITY OF AN EPA/NBS REFERENCE SLUDGE - TCLP LEACHATE,
SAMPLE NO. AIHL-85-0401
A. Results of Initial Screening Experiments
2 3
Extraction Residue Sample Specific Mutagenic Activity (revertants/mg) '
Zxp. Date Method^ Weight Volume TA98 TA100
(mg) (L) -S9 2%S9 10%S9 -S9 2%S9 10%S9
7/9/85 ^ Base/Acid 5.3 3 087 1287 1071 674 600 812
9/24/85 Base/Acid 12.1 6 990 1149 918 < 460 < 483 < 502
B. Comparison of Mutagenic Response in TA98 with 2% S-9 Mix
Exp. Date
Residue Sample Specific Mutagenic
Weight Volume Activity ^ 3
(mg) (L) (revertants/mg) '
Mutagenic Response
per Unit Volume
(revertants/L)
7/9/85 5.3 3 1287 2274
9/24/85 12.1 6 1149 2308
1. The extraction method indicates the order of pH in liquid-liquid extraction.
2. The specific activity of the mutagenicity dose-response curve is represented by
the slope (b as shown in the statistical analysis on graphs in Appendix O).
3. The spontaneous mutation and positive control values are listed on the Strain
Function Tests, Cell Titer and Viability Record in Appendix F.
4. The optimum testing condition in the initial screening experiment was in TA98
with 2% S-9 mix.
5. Activity in the background control (sodium acetate butter) was observed; the
screening experiment was repeated after making corrective actions.
-50-
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Table 13 lists the steps of investigation and the resulting change in the
mutagenicity comparison of water blank and sodium acetate buffer. An identical
experiment following the procedures of the initial experiment was performed to
confirm the existence of the problem. The sample preparation procedures involved
both TCLP and liquid-liquid extraction. The reagent ingredients for preparation of
the buffer were tested individually for mutagenicity. Negative responses were
observed for both chemicals. The extract of the buffer which was processed through
only the liquid-liquid extraction process was not mutagenic. The dichloromethane
used for the liquid-liquid extraction was concentrated, and the residue was tested
for mutagenicity. A negative response was obtained. Therefore, the mutagen was
not produced during the liquid-liquid extraction procedures.
As described in Section 5.1, Six Generic Sample Types, the TCLP involved
shaking, centrifugation, and filtration. Each step was evaluated separately. The
buffer extract which was processed without filtration exhibited an elevated but
below-thedetection-limit value. In contrast, the extract with the filtration produced
mutagenicity that was more than twice that of the background. The filtration
apparatus U9ed had a sinter-glass filter holder. It was suspected that the sinter-glass
was not clean because these glass particles can strongly adsorb potent mutagens
such as nitroarenes in a way that is similar to that shown by the affinity of silica
gel particles in a column. An all glass filtration apparatus with a stainless-steel
(inert) screen filter holder (SUPELCO, No. 5-8062) was replaced. This substitution
eliminated the mutagenicity problem.
In the meantime, a new recipe for the preparation of the sodium acetate
buffer was developed in a revised version of the TCLP method (revised on 10/4/85,
versus the previous draft on 3/2/85, see Section 5.1, Six Generic Sample Types, for
details). A new batch of buffer was prepared by combining 5.7 mL glacial acetic
acid and 64.3 mL IN sodium hydroxide solution which was then diluted to a volume
of 1 L. The pH was 4.93 + 0.02. The new buffer processed through the TCLP
procedures with the stainless-steel filter holder proved to be nonmutagenic.
A second screening experiment was performed on the TCLP leachate, and high
mutagenicity was observed again. Some priority pollutants were identified in the
leachate, and the concentrations were quantified. This information was provided by
-51-
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TABLE 13. INVESTIGATION OF THE MUTAGENICITY OF SODIUM ACETATE BUFFER USED
FOR THE TCLP LEACHATE PREPARATION
Exp. Date
Sample Preparation Procedures
Mutagenicity in TA98, -S9
(mean, revertants/plate)
Water Sodium Acetate
blank buffer
Shaking
TCLP
Centrifugation
Filtration
Liquid-
Liquid 2
Extraction
7/9/053
Yes
Yes
Yes6
Yes
36
n
CO
H
7/22/B53
Yes
Yes
Yes6
Yes
35
81
8/2/853'4
No
No
No
Yes
35
45
8/16/853
Yes
Yes
No
Yes
38
71
a/23/853
Yes
Yes
Ye36
Yes
30
80
9/24/B53
Yes
Yes
Yes7
Yes
37
54
11/22/85 ^
Yes
Yes
Yes7
Yes
38
68
1. The highest activity of the sodium acetate buffer was observed in TA98 without
S-9 in the initial screening experiment on 7/9/85. This condition was therefore
used for further investigation.
2. The dichloromethane residue was found to be nonmutagenic (See Appendix D).
3. The buffer was prepared by dissolving 8.2 g of anhydrous sodium acetate in 800
mL water by adjusting to pH 5 with glacial acetic acid and by diluting to a
volume of 1 L.
4. Both ingredients for preparation of the abovementioned buffer were found to be
nonmutagenic (See Appendix D).
5. The buffer was prepared by combining 5.7 mL glacial acetic acid and 64.3 mL
IN sodium hydroxide solution; dilution to a volume of 1 L followed. The pH
was 4.93 _~ 0.02.
6. An all glass filtration apparatus with a sinter-glass filter holder (MILLIPORE
No. XX15-04700) was used for filtration.
7. An all glass filtration apparatus with a stainless-steel screen filter holder
(SUPELCO No. 5-8062) was used for filtration.
-5?-
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Dr. L.R. Williams, EPA. A computer search by EMIC provided information on the
mutagenicity of these chemicals in the Ames assay. Table 14 lists the chemical
identities and their mutagenic activity and references.
Four chemicals were reported as mutagens: 3,3'-dichlorobenzidine, N-nitrosodi-
phenylamine, 2,6-dinitrotoluene, and acenaphthene. Reid et al. (1984), calculated
the activity in TA98 for 3,3'-dichlorobenzidine as 68 revertants/n mole which equaled
68 revertants/0.25 yg. The concentration of the compound in the leachate was 4.4
Ug/L. Therefore, the compound might produce approximately 1200 revertants/L
based on a simple computation. Approximately 2300 revertants/L were detected
(Table 12) in the leachate prepared for this project. Therefore, assuming no
complicated synergistic or antagonistic reactions occurred, 3,3'-dichlorobenzidine
accounts for approximately 52% of the activity in the leachate.
N-nitrosodiphenylamine and 2,6-dinitrotoluene are mainly active in TA100, and
their potencies are not as high as the 3,3'-dichlorobenzidine. A discrepancy on the
mutagenicity of the last chemical, acenaphthene, was found in the literature.
Gatehouse (1980), Hermann (1981), and others reported negative results, but Epler
et al. (1979), in citing results from other articles reported a positive result for the
chemical. Further evaluation is needed to differentiate the activity of the compound.
Possible Changes in Sample Composition—
In addition to the technical difficulties encountered during the liquid-liquid
extraction of the wastewater samples, some of the samples exhibited possible changes
in composition after storage at 4°C. The changes in composition were characterized
by any of the following: loss of mutagenic activity in the Ames assay; increase or
decrease in the amount of residue obtained after extraction; change in solubility
properties of the residue; change in odor; and change in appearance (color, particulate,
etc.). In all cases there was relatively no change in the sample pH after storage,
and there were no significant changes in the behavior of these samples during
processing.
Several of the samples exhibited a loss of mutagenic activity during storage.
The Stringfellow sample is a good example of this phenomenom (Figure 6). During
a period of only 13 days storage at 4°C (16 days after sample collection), the activity
-53-
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TABLE 14. MUTAGENICITY OF CHEMICALS IDENTIFIED IN THE EPA/NBS
REFERENCE SLUDGE - TCLP LEACHATE
Concentration
Mutagenicity
Chemical
in the
in the
Name
Leachate (yg/L)
Ames Assay
Reference(s)
diethylphthalate
7.1
Zeiger et al., 1985a
bis-2-ethylhe"xylphthalate
961
Zeiger et al., 1985a
butylbenzylphthalate
5.3
Zeiger et al., 1985a
di-n-butylphthalate
21
Zeiger et al., 1985a
di-n-octylphthalate
8.9
Zeiger et al., 1985a
1,4-dichlorobenzene
2
Haworth et al., 1983
acenaphthene
3.6
4*)
Gatehouse, 1980
(Epler et al., 1979)
acenaphthylene
0.89
.
Gatehouse, 1980
anthracene
6.2
-
McCann et al., 1975a
fluorene
5.3
-
McCann et al., 1975a
naphthalene
149
-
McCann et al., 1975a
2-methylnaphthalene
84
-
Hermann, 1981
phenanthrene
6.2
-
McCann et al., 1975a
dibenzofuran
5.3
-
Schoeny, 1982
nitrobenzene
5.3
-
Haworth et al., 1983
2,6-dinitrotoluene
16
+
Sandvall et al., 1984
3,3'-dichlorobenzidine
4.4
+
Reid et al., 1984
N-nitrosodiphenylamme
60
~
Haworth et al., 1983
-54-
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initially observed in the Ames a9say had been reduced by 50%. After almost three
months of storage, the mutagenic activity in this sample was no longer detectable,
and, in fact, the sample residue was highly toxic (See Appendix D).
Even more striking was the dramatic changes in the Stringfellow sample
appearance. After two months storage, the color had turned from light brown to
dark reddish brown and had become almost opaque. The odor was no longer sulfurou9,
and the sample had the aroma of an almond extract. The final residue obtained
after liquid-liquid extraction had an oily property that could not be evaporated to
complete dryness under nitrogen.
Similarly, the landfill surface runoff, exhibited loss of activity in the Ames
assay. The sample lost approximately 80% of its mutagenic activity in a period of
20 days, and, after 30 days, no mutagenic activity was detected; the residue was
extremely toxic to Salmonella typhimurium (Figure 7).
Not all samples exhibited changes in composition that were detected by the
Ames assay or that were observed during sample processing. The municipal water
effluent is an example of a highly stable wastewater in which the levels of mutagenic
activity after 74 days of storage were comparable to the results obtained after the
initial testing (Figure 8).
Because of the highly variable nature of these samples and because of the
absence of predictability, it was concluded that all processing of wastewater samples
should be completed within 14 days of receipt of the samples.
Liquid-Liquid Extraction
Samples Form Emulsions-
One common problem encountered by using the liquid-liquid extraction
procedure was the formation of an emulsion between the aqueous phase and the
organic phase during processing. The formation of an emulsion results in a less than
efficient separation of the two phases and in invariably poor recovery yields. This
problem is examplified by the municipal wastewater treatment plant effluent which
was characterized by a notable odor of raw sewage (Table 4). During extraction,
-56-
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125
LANDFILL SURFACE RUNOFF EXTRACT
100
(1)
¦w
rt
i—H
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os
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• 10/4/85
0 0.1
0.3 0.6
mg/plate
1.0
Figure 7. Dose-response curves showing degradation of mutagens in a
surface runoff sample from a class I hazardous waste
landfill.
-57-
-------
200
« 150
4->
ri
rH
Cu
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c
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50
MUNICIPAL WASTEWATER EFFLUENT
EXTRACT
A 8/2/85 Base/Acid
• 8/13/85 Base/Acid
¦ 10/17/85 Acid/Base
00.1 0.3 0.6 1.0 2.0
mg/Plate
Figure 8. Dose-response curves for a municipal wastewater treatment
plant effluent sample with stable mutagenicity.
-58-
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a heavy emulsion formed which was independent of the extraction pH. The best
separation of the aqueous-organic phases was accomplished by filtration through a
funnel packed with glass wool when the organic phase was eluted. In some cases,
a substantial quantity of the aqueous phase was included with the dichloromethane
eluent. The two phases were easily separated in a clean separatory funnel. The
resultant residue obtained from the liquid-liquid extraction of the municipal waste-
water effluent (9-17 mg/3 L) was characterized by a light brownish red color, that
dissolved readily in DMSO, and that exhibited significant mutagenic activity in the
Ames assay (See Section 6.1.1.3, Municipal Wastewater Treatment Plant Effluent,
for results). The addition of the glass wool filtration step was then included in the
general liquid-liquid extraction protocol for all samples that formed emulsions during
extractions.
Samples Form Precipitates-
Four of the six generic types of wastewater samples tested using the liquid-
liquid extraction procedure formed a substantial precipitate when the pH was raised
to a value approaching 11. This presented the most difficult problem associated
with this extraction technique. Several solutions were evaluated; this included:
changing the order of the pH extractions (i.e., acid extraction followed by the base
extraction); filtration; centrifugation before extraction; and centrifugation after
extraction. The following are examples of wastewater samples that formed a
precipitate during liquid-liquid extraction: (1) contaminated groundwater samples
from the Stnngfellow acid pits; (2) estaurine brackish surface water from the San
Francisco Bay and the brackish receiving water at the discharge site of an industrial
wastewater treatment plant; (3) surface runoff from a class I hazardous waste landfill;
and (4) TCLP extract leachate prepared from an EPA/NBS reference sludge. Those
samples could not be processed by following the suggested EPA protocol for the
preparation of wastewater exclusively. However, the inclusion of several steps was
found to increase the utility of this procedure. Since these four types of samples
precipitated only when the pH was raised to 11, it was logical to perform the first
extraction at pH = 2. Following this extraction, the pH of the sample was adjusted
to 11. The mixture was centrifuged, and the second extraction was performed on
the alkaline supernatant. In this way, the majority of the extraction will not be
affected by the precipitate since the mutagenic activity is predominantly extracted
during the initial extraction (See Section 6.1.3, Mutagen Extraction Efficiency, for
-59-
-------
recovery). In addition, if this procedure is followed, the difficulty in reconstituting
the sample (i.e., recombining the precipitate with the aqueous portion) can be avoided,
and a higher recovery would be expected.
The contaminated groundwater sample (Stringfellow) and the TCLP leachate
both exhibited a precipitate that partitioned with the organic phase. The Stringfellow
groundwater sample which was light brown in appearance and which was characterized
by an acidic pH (Table 4) exhibited a strong sulfurous odor. Analysis of this sample
revealed a high concentration of metal complexes (data not shown) that were insoluble
at pH > 3.2. Similarly, the NBS sludge sample contained high levels of metals.
Although the final residue weights of these two samples differed (Stringfellow
approximately 40-129 mg/3 L and TCLP approximately 5-6 mg/3 L), the residues
were soluble in DMSO, and they exhibited high mutagenic activity when applied to
the Ames assay.
The two San Francisco Bay brackish water samples and the landfill runoff
precipitated at pH 11, and this interfered with the extraction procedure. When the
revised protocol was followed, the residues were easily obtained, were readily dissolved
in DMSO, and both samples were found to be positive in the Ames as3ay.
Residual Water-
Bee ause of the persistence of the organic phase to retain trace amounts of
water, it was concluded that the anhydrous sodium sulfate column used before
evaporation was not adequate to completely remove all water. Residual water in
the concentrated extract and in vessels results in an incomplete evaporation process.
In addition, the residual water will greatly affect the final residue weight
determination used to calculate the final mutagenic potency of the sample. To
remove small quantities of residual water, this final 10 m!_ of extract was passed
over another sodium sulfate column (5 cm x 0.5 cm) before evaporation under
nitrogen. This step has been added to the general protocol as a precaution against
artifactual water contamination in the final residue.
Replace Kudema-Danish (K-D) Apparatus with Rotary Evaporator—
In the original consensus protocol, a K-D apparatus was recommended for
concentrating the extraction solvent, dichloromethane. However, dichloromethane
-60-
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has been reported as a Salmonella mutagen and a suspected animal and human
carcinogen. When the K-D concentrator is used, the solvent is heated and is vaporized
in the chemical fume hood. This practice causes release of the dichloromethane to
the general environment unless an activated-charcoal filter is placed properly at the
top of the venting systems above the hood to adsorb the solvent. Therefore, a
rotary evaporator was used as a concentrator instead of the K-D. The condensor
of the evaporator cools the dichloromethane vapor, and the used solvents are collected
in a waste reservoir. The solvent can then be treated as a carcinogen waste and
can be disposed of properly.
Mutagen Extraction Efficiency
Determination of the extraction efficiency for the recovery of various types
of mutagens from environmental water samples is critical in establishing a final
protocol because each laboratory that uses this method is required to operate a
formal QC program. The minimal requirements of this program include an initial
demonstration of laboratory capability and the analysis of spiked samples as a
continuing check on performance. Extraction efficiencies of the liquid-liquid extraction
protocol can be assessed either by instrumentation such as HPLC for analytical
identification and quantitation of the spiked standards or by the Ames assay for the
recovery of mutagenicity.
The efficiency of the liquid-liquid extraction protocol was determined by
measuring the recovery of three SPRM's in water. The three chemicals used in this
study, B(a)P, 2AA, and 4NBA, were separated and were quantitated by HPLC (See
Section 5.6.1, Chemical Analysis by HPLC, for details); they exhibited potent and
characteristic mutagenicity in the Ames assay. In addition, these compounds were
chosen because they represented three categories of chemicals; 4NBA represents an
acidic compound, B(a)P represents a neutral compound, and 2AA represents a basic
compound.
The overall efficiency of the liquid-liquid extraction protocol as outlined and
revised in the results section was calculated from data obtained from blank water
(ASTM Type I water) extraction. The recovery efficiencies for the three chemical
mutagens, added at various doses to blank water prior to extraction, are shown in
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Table 15. The recovery percentage was primarily calculated from HPLC analysis
of the residue obtained after extraction. However, for those doses where sufficient
amounts of the mutagen were used, the results obtained from the Ames assay were
compared to those obtained from HPLC analysis. The recovery of the neutral
compound, B(a)P, exhibited over 100% recovery at the highest spiked dose and fell
to less than 50% when the dose was less than 25 yg/1.5 L water. High doses of
2AA exhibited similar recovery efficiencies when compared to B(a)P (over 100%).
However, at doses less than 100 yg/1.5 L water, the efficiency decreased rapidly.
The recovery efficiency of 25 yg 2AA was approximately only one fifth of that for
B(a)P at the same dose. The recovery of the acidic compound, 4NBA, was not as
complete at the highest dose tested (approximately 60% to 80% recovery) when it
was compared with B(a)P or 2AA. In addition, a substantial decrease in recovery
efficiency for 4NBA was observed only when the dose (2.5 yg) was 2000-fold less
than the maximum dose (5000 yg). Also, the sequence of acid first followed by
the base extraction produced higher recovery (60% to 80%) than the recovery of
base first extraction (49% to 60%) in blank water.
These data suggest that low or trace levels of mutagens will be extracted
poorly relative to high levels of mutagens in an environmental sample. However,
because of the nature of this liquid-liquid extraction procedure extracting larger
quantities of an environmental wastewater sample to increase recovery efficiency
may not always be practical. Therefore, problems such as poor recovery efficiencies
of certain chemicals in the liquid-liquid extraction should be considered when assessing
unknown environmental complex samples to avoid underestimating the hazardous
potential.
The efficiency of the liquid-liquid extraction procedure for recovering these
three chemical mutagens was determined in a wastewater sample. The municipal
wastewater treatment effluent sample (No. AIHL-85-0403) was chosen because of its
stability and its representative behavior when processed by liquid-liquid extraction.
A maximum dose of each chemical mutagen was used to evaluate optimal conditions.
Two hundred fifty yg B(a)P and 2AA were added in 1.5 L of the water sample
whereas 5000 yg 4NBA was used because of the larger amounts needed for the
Ames assay. The percentage recovery of those three compounds is shown in Table
16. As expected from blank water efficiencies, B(a)P was recovered with
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TABLE 15. LIQUID-LIQUID EXTRACTION RECOVERY OF THREE SPIKED
REFERENCE MUTAGENS, BENZO(A)PYRENE (B(A)P), 2-AMINOANTHRA-
CENE (2AA), AND 4-NITROBENZOIC ACID (4NBA) ADDED TO LABORA-
TORY DISTILLED WATER
Mutagen
Extraction
Spiked Dose
"6 Recovery
Method^
Ames Assay HPLC
Added
(ug/1.5 L water)
-S9 2%S9 10%59
B(a)P
Base/Acid
500
.
79
185
110.
(neutral)
250
(pH
11 fraction)
68
131
85
250
(pH
2 fraction)
16
36
-
25
-
-
-
58
0.25
-
-
-
50
2AA
Base/Acid
500
121
119
105
(base)
250
(pH
11 fraction)
86
6 lx
80
250
(pH
2 fraction)
< 2
< kl
-
100
-
66
49
40
25
-
-
-
12
4NBA
Acid/Base
5000
80
m
60
(acid)
Base/Acid
5000
60,
-
-
i\ 9
2500
< 56
-
-
55
250
0
.
.
50
25
-
-
.
45
2.5
-
-
•
25
1. The extraction method indicates the order of pH in liquid-liquid extraction.
2. The number was calculated by using the method background value. The actual
plate counts were below the detection limit.
3. A combined extract of the pH 11 fraction and of the pH 2 fraction was analyzed.
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TABLE 16. LIQUID-LIQUID EXTRACTION RECOVERY OF THREE SPRVTS, B(A)P,
2AA, AND 4h«A ADDED TO THE MUNICIPAL WASTEWATER EFFLUENT
SAMPLE (NO. AIHL-85-0403)
Extraction
Mutagen
% Recovery
Method2
Added
Ames
HPLC
Base/Acid
0(a)P
90
103
2AA
353
29
4NBA
< 56 ,
Toxic
42
Acid/Base
B(a)P
105
100
2AA
27 3
25
4NBA
< 56
58
1. B(a)P or 2AA (250 ug/1.5 L wastewater, TA98, 10% S9 mix) or 4NBA (5000
ug/1.5 L wastewater, TA100, -S9) was added to a 1.5 L aliquot of the Municipal
Wastewater Sample, was extracted with dtchloromethane following the procedure
outlined in Section 5.4, and the recovery was analyzed by both the Ames assay
and HPLC.
2. The extraction method indicates the order of pH in the liquid-liquid extraction.
3. The number was calculated using the method background. The actual plate
counts were below the detection limit.
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approximately 100% efficiency regardless of the order of extraction (i.e., base first
or acid first). Surprisingly, the recovery of 2AA was significantly reduced to only
approximately 25% to 35% which is about half of the recovery efficiency in distilled
water. The order of extraction did not significantly affect the extraction efficiency.
The recovery of 4NBA in the Ames assay was incomplete due to the
below-the-detection-limit response, and the response was also below the maximum
60% to 80% recovery observed for blank water. The order of extraction may have
some effect on the recovery efficiencies of certain toxic chemicals since toxicity
was observed in the base first extract and not in the acid first extract. However,
the HPLC results indicated the 4NBA recovery in the municipal wastewater (42%
to 58%) was similar to that in blank water (49% to 60%). The sequence of acid
first when it is compared to the base first extraction effected an increase in recovery
from 60% to 80%.
It is concluded that the recovery percentage of a neutral compound such as
B(a)P, and maybe an acidic compound as 4NBA, is approximately the same in blank
water and in an environmental sample with the extraction characteristics of the
municipal wastewater effluent sample. The recovery of 2AA, a basic compound,
was substantially reduced in the municipal wastewater by using either the base first
or the acid first extraction scheme. Several possibilities may have contributed to
the lower extraction efficiencies of 2AA from the municipal wastewater sample when
it was compared to blank water; these include: chemical degradation; losses sustained
because of the formation of an emulsion; or loss of extraction efficiency as a result
of chemical-chemical interactions inherent in the sample (negative interference).
Further evaluation of the extraction efficiencies of various chemicals is needed to
verify the overall performance of this extraction procedure.
DEVELOPMENT OF QA PROGRAM
To ensure the production of data of continuing high validity, an intralaboratory
QA program has been developed.
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General Goal and Specific Objectives
The primary goal of establishing the QA program is to provide guidance to
ensure that all environmental sample preparation procedures and mutagenicity
measurements sponsored by EPA or other participating laboratories under regulations
such as the Toxic Substances Control Act (TSCA), the Federal Insecticide, Fungicide,
and Rodenticide Act (FIFRA), the Federal Food, Drug, and Cosmetic Act, and RCRA,
produce data of known quality. The quality of data is known and is reliable when
all components associated with its derivation are thoroughly documented and when
such documentation is both verifiable and defensible. The purpose of laboratory
analyses is to provide qualitative and quantitative data for use in decision-making
by management. The quality of the data must be specified, and sufficient resources
must be provided to assure that an adequate level of QA is performed. All tasks
should be undertaken with an adequate QA plan that specifies data quality goals
acceptable to the data user and that assigns responsibility for achieving these goals
(USEPA, 1984). In this fashion, the data will provide accurate and precise indications
for evaluating the situation and effect of the environment and will not lead to faulty
interpretation.
Specific objectives are as follows:
- To develop or put into service methods capable of meeting the users' needs for
precision, accuracy, sensitivity, and specificity.
- To establish the level of quality for routine performance of the laboratory and to
maintain a continuing assessment of the accuracy and the precision of analysts
within the laboratory group.
- To monitor the routine operational performance of the laboratory through an
appropriate intralaboratory program, to identify weak methodology, and to provide
sources for corrective actions as necessary.
- To detect training needs within the analytical group.
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- To provide a permanent record of instrument performance as a basts for validating
data and for projecting repair or replacement needs.
- To upgrade the overall quality of laboratory performance.
This program includes several key areas such as the periodic analysis and
interpretation of results of SPRM's, instrumental maintenance and calibration, and
monitoring of the quality of various reagents and solvents used in the sample
preparation and mutagenicity testing scheme.
The intralaboratory SPRM program provides a continuing measurement of the
performance capability of each analyst. Each person can be constantly aware of
his strengths and weaknesses, and corrective steps can be undertaken when necessary
before serious problems occur and before erroneous data are reported out of the
laboratory (Sherma, 1976).
Sufficient analyses are made on the unspiked subsamples so as to be satisfied
with the reproducibility of results from the same analyst and among all participating
analysts. When necessary, the sample may be sent to an outside laboratory (e.g.,
EPA-assigned reference laboratory) with experience in performing the analysis in
question for verification. A NBS standard complex mixture sample (e.g., NSS
reference sludge, TCLP leachate) with known stable chemical compositions may
be used to prepare the unspiked and spiked SPRM's. When reproducibility is
sufficient to establish a reliable mutagenicity profile in the unspiked sample, the
other half is spiked to produce residues with higher mutagenicity. The spiked
sample is thoroughly mixed, is transferred to small amber glass bottles with
Teflon-lined caps, and is stored in a freezer. These spiked samples serve to test
the capability of the analyst for recovery of higher mutagenicity levels. If the
compound(s) and media are known to be fully stable at room temperature or at
refrigerator temperature, freezer storage is not required.
For both the unspiked and spiked SPRM's, at least a dozen replications of the
analysis on the same sample should be conducted by chemists with recognized
competence. From this data, the percentage relative standard deviation is
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calculated and is U9ed in construction of control charts as described later in Section
6.2.3.2, QC Charts for Accuracy and Precision.
QC Approach
The approach to be implemented in order to achieve the objectives listed in
Section 6.2.1, General Goals and Specific Objectives, is recommended in this section.
These suggested policies (USEPA, 1979b) include:
- Publication, distribution, and maintenance of current and complete Laboratory
Sample Preparation and Mutagenicity Testing Methods and Procedures, Sample
Collection Information Record sheets, Strain Function, Cell Titer and Viability
Record sheets, QC Charts for Accuracy and Precision Measurements, Calibration
Data Sheets and Analytical Instrument Operating Instructions.
- Promulgation, distribution, and retention of laboratory reports with provision for
administrative/technical review.
- Periodic calibration of instruments and equipment, both in the laboratory and in
the field, QC checks on instruments for sample preparation and mutagenicity testing
procedures to ensure proper function at all times; and a preventive maintenance
program.
- Routine evaluations and maintenance of bacterial strains and the mammalian enzyme
systems with positive and negative controls.
- Assurance of appropriate, fresh reagents and chemicals and for appropriate,
calibrated glassware.
- Establishment and maintenance of total QC systems to assure continued precision
and accuracy of laboratory reports, including, as appropriate, requirements that:
a. Each procedure shall be checked on each day of use.
b. At least one standard (may be an instrument standard) and one control
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sample (working value established and run through the entire test
procedure) shall be included with each run of unknown samples. A
blank sample (no added amount of the constituent being determined)
may be a combination of field blank, travel blank, reagent blank, and
method blank; it shall be run to aid in detecting reagent contamination
and other problems important near the lower limit of ooeration of the
method.
c. If the results on the standard, control, or blank samples are not within
acceptable limits, the entire batch of analyses must be repeated and
control must be verified before reports are issued. Serious consideration
should be given to the nonacceptance of samples where there is only
enough material for a single analysis. There may be situations where
this policy is waived. Consideration of the consequences of reporting
results when the analytical system is apparently "out-of-control" should
minimize such waivers.
- Requirements for participation in interlaboratory QC evaluation programs.
- Requirements for training and qualifying personnel in QC techniques prior to running
new procedures. This qualification test is to be statistically valid and is to include
evaluation of precision and accuracy. The qualification standard shall be the
established level of quality of the laboratory.
Statistical QC Analyses and Records
There are two principal kinds of statistical tools available for use in QC
analyses: tests for differences including analysis of variance, and control charts.
For the protocol validation study, several statistical analyses were performed to
establish the method background; control charts for the background control were
then constructed for routine QC.
Method Background-
Since the mutagenicity results were used as the endpoint for data reporting
and evaluation, the protocol validation study involved both the sample preparation
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and the Ames testing procedures. Therefore, assuming that the basic variables such
as laboratory services, instrumentation, glassware, reagents, solvents, and gases etc.,
were under QC, the method background included three negative controls: the
spontaneous mutation revertants (SR) as the blank control for the mutagenicity
measurement of the bacterial strain, the dimethylsulfoxide (DMSO) revertants as the
solvent vehicle control in the Ames assay, and the revertants of blank water extract
representing the laboratory, field, travel, sample preparation method, and mutagenicity
procedure blanks.
Analysis of variance with two factor block design (« = 0.05) was used to test
the similarities among these three controls in TA98 under three S-9 conditions:
without, with 2% S-9 mix, and with 10% S-9 mix. Data from eight to nine experiments
on different dates were analyzed. Detailed calculations and primary data were listed
on the worksheet and were attached as Appendix G. The main effects of daily
variations and control conditions were tested using the F-test. If there was any
difference either among day-to-day or among three treatments, another statistical
analysis (t-test) was used to find out where the difference was (Dunn and Clark,
1974). Only TA98 data were evaluated because all samples contained mutagens
detectable in TA98 in this study and because the sample size for TA100 data were
too small for evaluation. This was the same reason that the data for TA98 with
30% S-9 mix were not analyzed.
The results in TA98 without S-9 showed that there was no difference among
daily measurements (p > 0.5), but there was significant difference among treatments
(p < 0.05). Data from each two controls were then compared by paired t-test («
= 0.05). Significant differences were found between SR and DMSO (p < 0.05),
between SR and water (p < 0.05), and between DMSO and water (p < 0.05). The
average plate count (revertants/plate) was 27 for DMSO, 30 for SR, and 35 for water
blank.
In the case of TA98 with 2% S-9 mix, the results showed that there was
significant difference either among daily measurements or among three treatments
(p < 0.05). The differences may be attributed to unstable data that results from
combinations of variation in bacteria, in testing conditions, and/or in preparation
procedures. The t-test comparison indicates similarity between SR and DMSO (p >
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-------
0.5), and significant difference between SR and water (p < 0.05) and between DMSO
and water (p < 0.05). The average plate count (revertants/plate) was 40 for DMSO,
44 for SR, and 49 for water blank.
In TA98 with 10% S-9 mix, the results indicated that there was no significant
difference among daily measurements (p > 0.5), but there was a significant difference
among three controls (p < 0.05). Again, the t-test results showed similarity between
SR and DMSO (p > 0.05), and significant difference between SR and water (p < 0.05)
and between DMSO and water (p < 0.05). The average plate count (revertants/plate)
was 44 for DMSO, 45 for SR, and 51 for water blank.
In conclusion, the three types of controls were different from each other in
most of the cases. The SR and DMSO values could not be included as part of the
method background. The water blank values including the effect of the laboratory
process, field and travel procedure, sample preparation method, and mutagenicity
testing, were accepted as the method background. The value of method background
was 35 (without S-9), 49 (2% S-9), 51 (10% S-9), and 39 (30% S-9). The detection
limit for the Ames assay, which was twice the background, as discussed previously
in Section 6.1.1, Ames Assay Results and Interpretation, would be twice that of the
water blank value.
QC Charts for Accuracy and Precision—
In order to establish the method background, valid precision and accuracy data
must be developed in addition to the abovementioned statistical analyses. Accuracy
and precision are two aspects of the possible error of every scientific measurement.
Accuracy relates to the closeness of approach of a single measurement, or
of the average of a series of measurements, to the true value. The true value is
incapable of being measured exactly, but in many cases it can be estimated very
closely. By using calibrated equipment, by performing the work extremely carefully,
by executing a very large number of measurements, and then by applying statistics
to the results, reasonable approximations of the true value can be obtained.
Frequently, the true value is estimated from the results of different analysts in
different laboratories. Precision describes the closeness of approach of replicate
results to a common value. Repeated measurements of the same quantity will usually
-71-
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not be identical but will scatter around some common value. Precision describes
the reproducibility or the scatter of a series of measurements or results.
One commonly used method for analyzing potential errors of any measurement
is the construction of tion of Quality Control Charts. That is, in addition to recording
numerical results of each analysis, the result is plotted as a point in a chronological
sequence on a chart.
The purpose of this chart is to provide graphic assessment of accuracy and
precision for each analysis and to provide instant detection of erroneous data. The
charts allow quick observation of trends for a particular analysis and have long term
value for the self evaluation of analytical output by staff personnel. Another
significant value of the charts is that of providing a laboratory administrator with
a rapid assessment of the continuing analytical capability of the staff scientists as
related to the output of valid analytical data. The essential elements common to
control charts include an expected value (the central line) and an acceptable range
of occurrence (the region between upper and lower control limits), as shown in
Figures 9, 10, and 11 (Software Consulting Group, 1984).
Figures 9, 10, and 11 are QC charts of the method background (i.e., water
blank control) in TA98 in the absence and in the presence of 2% and 10% S-9 mix.
Nine to 11 sets of data were collected from different experiments in this project
and were used for construction of these QC charts.
The upper graphs represent the QC charts for accuracy measurements.
Accuracy is the difference between a measurement and a true value. The calculated
mean value (XBAR, or =) was assumed as the true value. The upper control limit
(UCL^) and lower control value (LCL^) for the 99% confidence interval was then
computed. The calculation process was recorded on the worksheets attached as
Appendix H. The control chart is then the graphical presentation of the experimental
results plotted in relation to these calculated limits. If the results fall within the
limits, the experimental process is considered "in control." Otherwise, the process
is judged as "out of control."
-72-
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Figure 9. Control charts of the method background in TA98 without S-9.
-73-
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Control charts of the method background in TA98 with 2% S-9 mix
Figure 10
-------
QC ACCURACY TA90 10Z S9 WATER
UCL(XBAR)
MEAN (XBAR)
LCL(XBAR)
SUBGROUP NUMBER
QC PRECISION TA90 10* S9 WATER
28
23
17
11
5
-001,
*
UCL(R)
X
—i—
in
MEAN
-------
The lower graphs represent the QC charts for precision measurements.
Precision is a measure of reproducibility of results. It can be expressed as SD,
variance (V), or range (R). When the number of replicates (n) is small as in this
project (n = 9-11), the range chart is the most efficient way to measure precision.
Range is the difference between the highest and the lowest measurements. The
upper control limit (UCL^) and lower control limit (LCL^) were computed (Appendix
H). Again, 99% of the calculated range values are expected to be within the
boundary of these control limits. If eight or more successive values appear on one
side of the mean value line, the process is considered as biased. The source of
error which causes the bias should be traced. If the experimental results fall outside
of the control limits, the process is judged as "out of control."
Graphs on Figures 9, 10, and 11 indicate that the water blank values in all
three conditions are all within the control limits of accuracy and precision
measurements. Continuation of these analyses and systematic routine evaluations
will establish a large data base and will ensure the validity of the sample preparation
and of the mutagenicity testing procedures.
The upper limit for accuracy of the method background was 44 (without S-9),
62 (2% S-9), and 62 (10% S-9). The lower limit was 26 (without S-9), 33 (2% S-9),
and 39 (10% S-9). The range for precision of the method background was 0-23
(without S-9), 0-37 (2% S-9), and 0-28 (10% S-9).
Sample sizes are related to the number of replications. Since triplicate plate
counts are used as a single control chart point, the chances of picking up small
changes in the process average are increased. The protection against not detecting
small changes in the process increases as the sample size increases. The use of
frequent sampling will detect changes more quickly with time. The ultimate goal
would be large sample sizes and measurements taken frequently. However, an
economic decision has to be made as to the value of closer control versus the
increased cost of attaining that degree of control.
In environmental measurements, separate control charts are recommended for
each parameter, for each instrument, and for each analyst. However, the true value
of the investigated parameter may vary considerably among samples. This variability
-76-
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in true value means there are no expected numbers for randomly selected samples
so that the accuracy of testing methodology must be evaluated indirectly through
the recovery of standards and of spikes as described previously in Sections 5.6.2,
Mutagenicity Recovery Analysis, and 6.1.3, Mutagen Extraction Efficiency.
Once the valid precision and accuracy data are developed for each method
and each analyst, to insure that valid data continue to be produced, systematic
routine checks must show that the test results remain reproducible and that the
methodology is actually measuring the quantity in each sample. In addition, QC
must begin with sample collection and must not end until the resulting data have
been reported. QC of analytical performance within the laboratory is thus but one
vital link in the dissemination of valid data to the public. Understanding and
conscientious use of QC among all field sampling personnel, analytical personnel,
and management personnel is imperative to the success of this project.
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Acad. Sci. USA 72:979-983.
McCann J, Spingarn NE, Kobori J, Ames BN. 1975b. Detection of Carcinogens as
Mutagens: Bacterial Tester Strain with R Factor Plasmids. Proc. Natl. Acad. Sci.
USA 72:979-983.
Metcalfe CD, Sonstegard RA, Quilliam MA. 1985. Genotoxic Activity of Particulate
Material in Petroleum Refinery Effluents. Bull. Environ. Contam. Toxicol. 35:240-248.
Mortelmans K, Stocker BAD. 1979. Segregation of the Mutator Property of Plasmid
R46 from Its Ultraviolet-Protecting Property. Mol. Gen. Genet. 167:317-327.
Nichols FH, Cloem JE, Luoma SN, Peterson DH. 1986. The Modification of an
Estuary. Science 231:567-573.
Reid, TM, Wang CY, King CM, Morton KC. 1984. Mutagenicity of Some Benzidine
Congeners and Their N-Acetylated and NjN'-Diacetylated Derivatives in Different
Strains of Salmonella typhimurium. Environ. Mutagen. 6:145-151.
Sandvall A, Marklund H, Rannug U. 1984. The Mutagenicity of Salmonella
typhimurium of Nitrobenzoic Acids and Other Wastewater Components Generated in
the Production of Nitrobenzoic Acids and Nitrotoluene. Mutat. Res. 137:71-78.
Schoeny R. 1982. Mutagenicity Testing of Chlorinated Biphenyls and Chlorinated
Dibenzofurans. Mutat. Res. 101:45-56.
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Sexton N, Myers L, Hughes T. 1981. A Plan to Develop and Implement a Quality
Assurance Program for the Ames/Salmonella Test. EPA-600/2-81-054. Health Effects
Research Laboratory, USEPA, Research Triangle Park, NC.
Sherma J. 1976. Manual of Analytical Quality Control for Pesticides and Related
Compounds in Human and Environmental Samples. EPA-600/1-76-017. Health Effects
Research Laboratory, USEPA, Research Triangle Park, NC.
Shokes RF. 1984. Progress Report. Stringfellow Facility Remedial Investigation/
Feasibility Study. Task III: August, 1984 Groundwater Monitoring Survey. Preoared
by JRB/5AIC for Division of Toxic Substances Control, CDHS, Sacramento, CA.
Software Consulting Group. 1984. Application Program: X,R,P and C Control
Charts, User's Manual, Revision 2. SCG, P.O. Box 3298, Santa Clara, CA.
Sugimura T, Nagao M. 1980. Modification of Mutagenic Activity. In: Chemical
Mutagens, Vol. 6, pp. 41-66. FJ deSerres, A Hollaender, ed. Plenum Press, New
York.
Sugimura T, Nagao M. 1982. The Use of Mutagenicity to Evaluate Carcinogenic
Hazards in Our Daily Lives. In: Mutagenicity: New Horizons in Genetic Toxicology,
pp. 73-88. J. Heddle, ed. Academic Press, New York, NY.
USEPA. 1979a. Hazardous Waste Fact Sheet. EPA Journal. February 1979.
USEPA. 1979b. Methods for Chemical Analysis of Water and Wastes. EPA-600/4-79-
020. Environmental Monitoring and Support Laboratory, USEPA, Cincinnati, OH.
USEPA. 1982. Test Methods: Test Methods for Evaluating Solid Waste, Physical/
Chemical Methods, SW-846. Second edition. Office of Solid Wastes, USEPA,
Cincinnati, OH.
USEPA. 1984. Guidance for Preparation of Combined Work/Quality Assurance
Project Plans for Environmental Monitoring. (OWRS QA-1). Office of Water
Regulations and Standards, USEPA, Washington, D.C.
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Williams LR, Preston JE. 1983. Interim Procedure for Conducting the Salmonella/
Microsomal Mutagenicity Assay (Ames Test). EPA-600/4-82-068. Environmental
Monitoring Systems Laboratory, USEPA, Las Vegas, NV.
Williams LR. 1985. Quality Assurance Considerations in Conducting the Ames Test.
In: Quality Assurance for Environmental Measurements, pp. 260-291. JK Taylor,
TW Stanley, ed. ASTM Special Technical Publication 867. ASTM, Philadelphia, PA.
Zeiger E, Pagano DA, Robertson IGC. 1981. A Rapid and Sample Scheme for
Confirmationn of Salmonella Tester Strain Phenotype. Environ. Mutagen. 3:205-209.
Zeiger E, Haworth S, Mortelmans K, Speck W. 1985a. Mutagenicity Testing of
Di(2-ethylhexyl)phthalate and Related Chemicals in Salmonella. Environ. Mutagen.
7:213-232.
Zeiger E, Risko KJ, Margolin BH. 1985b. Strategies to Reduce the Cost of
Mutagenicity Screening with the Salmonella Assay. Environ. Mutagen. 7:901-911.
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APPENDIX A
ORIGINAL PROTOCOL FOR ENVIRONMENTAL WATERS AND WASTEWATER
The original consensus protocol for preparing environmental water and wastewater
samples, published as part of the meeting proceedings (ICAIR ed., 1985), is attached
in this report as Appendix A for detail descriptions of the samle preparation procedures.
PROTOCOL FOR THE PREPARATION OF ENVIRONMENTAL WATERS AND WASTEWATER FOR
MUTAGENICITY TESTING
1.0 Scope and Application
1.1 This is a general purpose method which covers che preparation of waste-
water samples for solvent-extractable organic compounds chat are amenable to
Ames mutagenicity assay.
1.2 The method is applicable only to solvent-excraccable organic compounds,
and not all chemicals in che sample are stable or recoverable by this
procedure.
1.3 The final extract will be subjected to the Ames mutagenicity assay using
the procedure of Williams and Preston (1982).
2.0 Summary of Method
Cenotoxic activity of wastewater samples is assessed on the liquid and
solid phases of wastewater grab samples. The discrete phases are collected,
quantified, extracted and concentrated, and the resultant concentrates are
transferred to DMSO and subjected to bioassay evaluation using a standard Ames
test (Ames et al. 1975, Brusick and Young 1981, Williams and Preston 1982).
3.0 Definitions
Environmental Waters and Wastewater - Water not intended for human
consumption, containing less than 502 suspended solids by weight, including
water from industrial emissions, rivers, lakes and ponds. Wastewater may
contain nonaqueous liquids in addition to the aqueous and solid phases.
Internal Standard - A pure compound added to a sample in known amounts
and used to calibrate concentration measurements of other compounds that are
sample components.
Field Duplicates - Two samples taken at the same time, placed under
identical circumstances and treated exactly the same throughout field and
laboratory procedures. Analysis of field duplicates indicates the precision
associated with sample collection, preservation and storage, as well as with
laboratory procedures.
Reagent Blank - Reagent water placed in a sample container in the
laboratory and treated as a sample in all respects, including exposure to
sampling site conditions, storage, preservation and all analytical procedures.
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Nonaqueous Liquids Phase - Liquids whose major component is not vater
and vhich form discrete zones in an aqueous medium.
Sediment Solid Phase - The solid or semisolid phase recovered from a
24-h resting wastewater sample.
<>.0 Interferences
4.1 Samples may change or become contaminated during transport from the
collection site to the laboratory. Sample custody and shipping/storage
conditions must be fully described and documented.
4.2 Emissions from wastewater sources change with time. Sufficient sample
to complete all phases of the evaluation should be collected at the time of
sampling. Date, time and exact location of sampling must be fully described
and documented.
4.3 Method interferences may be caused bv contaminants in solvents,
reagents, glassware and other sample processing hardware that lead to discrete
artifacts and/or elevated baselines in the total ion current profiles. All of
these materials must be routinely demonstrated to be free from interferences
under the conditions of the analysis by running laboratory reager.t blanks.
4.i* Matrix interferences may be caused by contaminants that are coextractec
from the sample. The extent of matrix interferences will vary considerably
from source to source, depending upon the nature and diversity of the in-
dustrial complex or municipality being sampled. An internal standard shculd
be employed to evaluate matrix interference.
4.5 Methylene chloride can interfere with the mutagenicity assay, so care
must be exercised to completelv remove all methylene chloride prior to
dilution with dimethyl sulfoxide.
4.6 The sample vill be supplied for Ames testing in its most concentrated
fore in dimethyl sulfoxide, because additional sample concentration in the
presence of dimethyl sulfoxide (B.P. .189 C) is prohibitive due to potential
thermal or evaporative loss of sample organic constituents. If the extract is
not soluble in its concentrated form, dilution with additional dimethyl
sulfoxide or other appropriate solvents is advised.
4.7 Some extracted concentrated samples in DKSO may be highly cytotoxic in
the Ames test. If a sample cannot be tested up to a level of 1,000 yg ex-
tracted organics per plate, it should be considered "toxic," and altem?tive
treatment or bioassay methods should be performed, including chromatographic
separation of toxic compounds from nontoxic compounds (HPLC or acid/base/
neutral fractionation) (Hughes et al. 1980, Tabor and Loper 1980). In the
case of limited sample size, the maximum level will depend only on sample
availability.
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5.0 Safety
5.1 The toxicity or carcinogenicity of each reagent used in this method has
not been precisely defined; however, each chemical compound should be treated
as a potential health hazard. From this viewpoint, exposure to these
chemicals must be reduced to the lowest possible level by whatever means
available.
5.2 The laboratory is responsible for maintaining a current awareness file
of OSHA regulations regarding the safe handling of the chemicals specified in
this method (NIOSH 1977, OSHA 1976). A reference file of material data
handling sheets should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are available
and have been Identified for the information of the analyst (ACS 1979).
5.3 Solvent rinsing of vessels with methylene chloride may result in
excessive exposure to this solvent. All rinsing should be performed in an
approved chemical fume hood, and appropriate personal safety apparel should be
worn.
5.<* Internal control samples may be spiked with chemicals demonstrating
carcinogenic activity in animals. Internal control samples should be handled
according to safety procedures consistent with this fact (NCI 1981).
5.5 Care should be used when pressurizing or vacuum-filtering samples.
Excessive pressure or vacuum might cause vessel or filter breakage and
expulsion of concentrated sample into the laboratory environment.
5.6 Since some wastewater samples may come from sources containing human or
domesticated animal excrement, communicable disease risk is present. Labora-
tory personnel should be adequately immunized for communicable diseases, and
all samples should be considered potentially contaminated and handled accord-
ingly.
6.0 Apparatus and Equipment
6.1 Grab Sample Bottle-
Amber glass wide-mouth, 3.8-L or 1-gal volume, fitted with screw caps
lined with Teflon. Aluminum foil may be substituted for Teflon if the sample
is not corrosive. Alternatively, Amicon stainless steel 20-L carboys may be
used (see Drinking Water Protocol).
6.2 Pressure Filter—
Millipore unit fitted with a glass fiber filter (1-u pore size).
6.3 Centrifuge—
High-speed, refrigerated sample centrifuge capable of handling 50C-ml
centrifuge tubes.
6.4 Celite column to trap particulate matter prior to resin concentration
(acceptable devices are comrercially available).
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6.5 Glassware (all specifications are suggested. Catalog numbers are
included for illustration only).
6.5.1 Separatory funnel—2,000 ml, with Teflon stopcock.
6.5.2 Drying column—9-mo ID chromatographic column with coarse frit.
6.5.3 Concentrator tube, Kuderna-Danish—25 mL, graduated (Kontes
K-570050-2525 or equivalent). Calibration must be checked at the volumes
employed in the test. Ground glass stopper is used to prevent evaporation of
extracts.
6.5.<* Evaporative flask, Kuderna-Danish—500 mL (Kontes K-570001-0500 or
equivalent). Attach to concentrator tube with springs.
6.5.5 Snyder column, Kuderna-Danish—Three-ball macro (Kontes K—503000—0121
or equivalent).
6.5.6 Vials—Amber glass, 10- to 5-mL capacity, with Teflon-lined screw cap.
6.5.7 Continuous liquid-liquid extractors—Equipped with Teflon or glass
connecting joints and stopcocks requiring no lubrication. (Rershberg-Wolf
Extractor-Ace Glass Company, Vineland, N.J. P/N 6B4-0 or equivalent).
6.6 Boiling Chips—
Approximately 10/40 mesh. Heat to 400 C for 30 min or Soxhlet extract
with methylene chloride.
6.7 Water Bath—
Heated, with concentric ring cover, capable of temperature control
(i 2 C). The bath should be used in a chemical fume hood.
6.8 Balance—
Analytical, capable of accurately weighing 0.0001 g.
6.9 Nitrogen Evaporator—
Equipped with Teflon or glass jets and temperature control. (Meyer
N-evap Model 112 or equivalent - Organomation Assoc., Inc., Northborough, MA).
6.10 Standard amber glass storage containers, 10-mL bottles with
Teflon-lined screw caps.
7.0 Reagents and Consumable Materials
7.1 Reagent Water—
Reagent water is defined as a water in which an interferent is not
observed at the method detection limit of each parameter of interest. It
consists of XAD-2 cleaned tap water.
7.2 Sodium Hydroxide Solution (10 N)—
Dissolve 40 g NaOH in reagent water and dilute to 100 mL.
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7.3 Sodium Thiosulfate (ACS), granular.
7.4 Sulfuric Acid Solution (1*1) —
Slowly add 50 mL of H^SO^ (sp. gr. 1.84) to 50 mL of reagent water.
7.5 Acetone, methanol, methylene chloride, dimethyl sulfoxide (pesticide-
quality or equivalent).
7.6 Sodium Sulfate (ACS) granular, anhydrous—
Purify by heating at 400 C for 4 h in a shallow tray.
7.7 Internal Standard Mutagen—
Use 4-nitroquinoline-N-oxide dissolved at 100 ug/L of liquid sample or
other acceptable mutagens.
8.0 Quality Control
8.1 Each laboratory that uses this method is required to operate a formal
quality control program. The minimum requirements cf this program consist of
an initial demonstration of laboratory capability and the analysis of spiked
samples as a continuing check on performance. The laboratory is required to
maintain performance records to define the quality of data that is generated.
Ongoing performance checks nust be compared with established performance
criteria to determine if the results of analyses are within accuracy and
precision limits expected of the method.
8.1.1 Before performing any analyses, the analyst must demonstrate the
ability to generate acceptable accuracy and precision with this method.
8.1.2 In recognition of the rapid advances that are occurring in chroma-
tography, the analyst is permitted certain options to improve the separations
or lower the cost of measurements. Each time such modifications are maae to
the method, the analyst is required to repeat the procedure in Section 8.2.
8.2 To establish the ability to generate acceptable accuracy and precisior,
the analyst must perform the following operations.
8.2.1 Select a representative spike concentration for each parameter to be
measured. Using stock standards, prepare a quality control check sample
concentrate in acetone 1,000 times more concentrated than the selected concen-
trations. Quality control check sample concentrates, appropriate for use with
this method, will be available from the U.S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268.
8.2.2 Using a pipet, add 1.00 mL of the check sample concentrate to each cf a
minimum of four 1,000-oL aliquots of reagent water. A representative waste-
water should be used in place of the reagent water, but one or more additional
aliquots must be analyzed to determine background levels, and the spike level
must exceed twice the background level for the test to be valid. Analyze the
aliquots.
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8.3 It Is recommended chat the laboratory adopt additional quality
assurance practices for use with this method. The specific practices that are
most productive depend upon the needs of the laboratory and the nature of the
samples. Field duplicates may be analyzed to monitor the precision of the
sampling technique. Whenever possible, the laboratory should perform analysis
of standard reference materials and participate in relevant performance
evaluation studies.
9.0 Procedure
9.1 Sample Collection—
9.1.1 Wastewater collection methods—It is recommended that samples be taker,
which reflect the "normal" state of the sample site. Periodic sampling of the
same site over several months may also be valuable in providing information on
peak periods of activity. For aqueous samples, the most common sampling
procedure is a manual grab collection of the volume needed for analysis.
Once collected, samples should be placed in the sealed amber glass bottles and
held at 4 C during any storage or shipping. The head space in the container
should be reduced by complete filling of the container or by replacement with
a N, blanket. Table 5 identifies the volume of sample required.
9.1.2 Wastewater sample custody—An example of a sample custody form is shown
in Figure 12. This form is from EPA document EPA-600/881-024 (Brusick and
Young 1981). Either this EPA form or its equivalent should be prepared at the
tine the sample is collected, and a copy of the form should accompany the
sample through all phases of processing and bioassay.
9.1.3 Storage and use of sample—It is strongly recommended that all samples
be processed (separated, extracted and concentrated) within 14 days after
collection and completely analyzed within 40 days of concentration and solvent
exchange.
9.2 Sample Separation into Component Phases—
9.2.1 Each sample container is stored motionless at a C for 2u h after
receipt. At this point, a gravity separation of phases will occur. Any
nonaqueous liquid phases identified in the liquid component of the sample will
be separated from the containers, combined into one sample and processed as a
nonaqueous liquid waste.
Any solid sediment on the bottom of the sample container will be
collected, combined into a single sample and processed as a waste solid.
The aqueous phase, with or without suspended particles will be
recovered and treated in one of three methods (see Section 9.2.3).
9.2.2 Each liquid phase recovered must be weighed to the nearest gram and the
volume measured. The solid sediment is weighed (net weight to the nearest
gram). Like phases are combined into a common vessel for processing. Once
combined, total weights and/or volumes are calculated. Storage conditions are
the same as defined for the initial collected sample. No attempts to control
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TABLE 5.
RECOMMENDED VOLUMES AND
STORAGE OF SAMPLES
Component
Volume/Weight
Storage Conditions
Collected Sample
Minimum of 30 L
A C, dark
Gravity Separation
Method
Minimum of 30 L
4 C, dark motionless
Sample for Resin
Column Concentration
10 L
4 C, dark glass or
Teflon-lined vessel
Liquid/Liquid Extrac-
tion Method
3 L
4 C, dark glass or
Teflon-lined vessel
Solids for Solid Waste
Extraction
500 g net weight
<* C, dark closed con-
tainer
Extracted/Concentrated/
Solvent Exchanged
Sample for Bioassay
10 nL
4 C, amber glass vial
with Teflon cap liner
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I. SAMPLE INFORMATION
1. Sample No. Collection Date
fa)
2. Sampling Site
3. Field Sampling Manager (on-site)
4. Contractor Contract No.
5. EPA Project Officer Program Name
6. Source Sampled
7. Discharge Rate of Source (Volume/Time)
8. Quantity Sampled/Units
9. Sample Description (liquid, slurry, solid, extract, appearance, etc.)
10. Other Information as Applicable
Collection temp.
PH
Other
11. HANDLING & SHIPPING
1. Describe Sample Treatment Prior to Shipping (e.g., transfers,
extractants, stored undiluted, grinding, solvents used)
2. Field Storage and Shipping Conditions
Container Temperature Light
Ambient Shield from light
Refrigerate (0 to <* C)
Freeze (-20 C)
Dry Ice
3. Approximate Time in Storage and Time in Shipping
4. Sample Shipped to
5. Mode and Carrier for Shipping
6. Comments
(This form should be completed by the on-site sampling manager and accompany
each sample.)
(a) Graphically illustrate site of collection.
Figure 12. Sample information.
(Extracted from: IERL-RTP Procedures Manual Level I Environmental Assessment)
Biological Tests EPA-600/8-81-024, October, 1981. pp. 138.)
Sampling location
Sampling technique
Amber Glass
Polyethylene Bottle
Coated Bag or Bottle
Teflon or Tedlar Bags
Other
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pH or change the pH of Che sample are required. The initial sample containers
should be rinsed three times with methylene chloride. The final rinse collec-
tion will be concentrated and added to the final extract sample prior to
solvent exchange.
9.2.3 The liquid phase recovered from the sample will be processed by one of
the following methods:
a. If the sample has <57. suspended solids by weight (reference for
method TSS), the sample may be extracted and concentrated by the
techniques described in the Drinking Water Protocol, with the
addition of ASTM Celite prefliter to the concentrator apparatus.
b. If the sample has >5% suspended solids by weight, the sample stay
be further separated by high-pressure filtration or high-speed
centrifugation into liquid (<5Z suspended solids) and solids.
These two phases can be processed further by the Drinking Water
Protocol and Solid Waste Protocol, respectively.
c. If the sample has >5% suspended solids by weight, it may be
processed by a liquid/liquid extraction method (Section 9.3). If
the bioassay from the sample recovered from this method is nega-
tive, consideration should be given to processing a retained
liquid phase (10 L) by the method described in Section 9.2.3.b.
9.3 Separators Funnel Liquid-Liquid Extraction Method—
9.3.1 Shake the sample, contained in a gallon amber glass bottle, to assure
homogeneity. Samples are usually extracted using separatory funnel tech-
niques. If emulsions will prevent achieving acceptable solvent recovery with
separatory funnel extractions, continuous extraction may be used.
9.3.2 Measure two 1,500-aL sample allquots into two 2000-mL separatory
funnels. Check the pH of the sample with wide-range pH paper and adjust to
pH 11 with ION sodium hydroxide.
9.3.3 Add 150 mL of methylene chloride to each separatory funnel, and extract
the sample by shaking the funnels for two minutes with periodic venting to
release excess pressure. Allow the organic layers to separate from the vater
phases for a minimum of ten minutes If the emulsion interface between layers
is more than one-third the volume of the solvent layer, the analyst must
employ mechanical techniques to complete the phase separation. The optimum
technique depends upon the sample, but may including stirring, filtration of
the emulsion through glass wool, centrifugation or other physical nethods.
Combine the methylene chloride extracts in a 1,000-oL Erlenneyer flask. If
the emulsion cannoc be broken (recovery of less than 80% of the methylene
chloride, corrected for the water solubility of methylene chloride), transfer
Che sample, solvent and emulsion into the extraction chamber of a continuous
extractor and proceed as described in Section 10.
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9.3.4 Add 100 mL of methylene chloride to each separatory funnel, and repeat
the extraction procedure a second time, combining all extracts in the 1,000-raL
Erlenmeyer flask. Perform another 100-mL extraction in the same manner.
9.3.5 Adjust the pH of the aqueous phases to less than 2 using the sulfuric
acid solution. Serially extract the sample with 150, 100 and 100 mL of
methylene chloride per separatory funnel. Combine the extracts with previous
extracts in the 1,000-mL Erlenmeyer flask. Notice that additional
fractionation could be achieved here, if_ requested, by not combining these
extracts (organic acids) with previous extracts (organic base/neutrals) but,
rather, processing each separately through the remaining procedure.
9.3.6 Assemble a Kuderaa-Danish (K-D) concentrator by attaching a 25-nL
concentrator tube to a 500-mL evaporative flask. Other concentration devices
or techniques may be used in place of the Kuderaa-Danish. Pour one half of
the combined extract through a drying column containing about 10 cm of
anhydrous sodium sulfate, and collect the extract in the K-D concentrator.
Add one or two clean boiling chips and attach a three-ball Snyder column.
Prewet the Snyder column by adding about 1 mL of methylene chloride to the top
of the column. Place the K-D apparatus on a hot water bath (60 to 65 C) so
that the concentrator tube is partially immersed in the hot water, and the
entire lower rounded surface of the flask is bathed with hot vapor. Adjust
the vertical position of the apparatus and che water temperature as required
to complece the concentration in 15 to 20 minutes. At the proper rate of
distillation, the balls of the column will actively chatter, but the chamoers
will not flood with condensed solvent.
9.3.7 When the apparent volume of liquid reaches approximately 10 mL, remove
the K-D apparatus from the water bath and add the remaining combined extract
through the drying column and into the K-D apparatus. Rinse the Erlenmeyer
flask and column with 25 mL of methylene chloride to complete the quantitative
transfer. Add two clean boiling chips and continue concentration on the water
bath until the liquid reaches 10 mL. Remove the K-D apparatus from the water
bath and allow it to drain and cool for at least 10 minutes. Remove the
Snyder column and rinse the flask and its lower joint into the concentrator
tube with 1 to 2 mL of methylene chloride.
9.3.8 Concentrate the methylene chloride extract to 10 mL using the nitrogen
evaporator. Do not allow the bath temperature to exceed 35 C. An aliquot
should be removed for total organic carbon (T0C) analysis (Lentzen et al.
1978). If requested, at this point a 1-mL aliquot may be removed for anslvsis
by gas chromatograpn/mass spectrometer using Method 625 (44 FR 233, December 3,
1979). Transfer the extract to a tared, 3-dram glass vial that is etched at
the 5-mL mark. Weigh the vial and its contents and record the weight of the
residue. Dilute the sample extract to the 10-mL mark with dimethyl sulfoxide
and attach the Teflon-lined screw cap to the vial. From the T0C determination,
the DKSO extract should be recorded as mg organics/mL DMS0. Refrigerate the
extract until ready for Ames testing.
9.3.9 If no aliquot was removed for analysis by gas chromatograph/mass
spectrometer, the DMSO extract represents a concentration factor of
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approximately 600x. If an aliquot was removed for analysis by gas chroma-
tograph/mass spectrometer, Che DMSO extract represents a concentration factor
of approximately 540x.
10.0 Calculations
All measurements of mutagenic activity should be expressed as revertants per
liter of emission and as a rate {revertants/liter/hour). In order to generate
these values, data for revertants/mg organics extracted by each sample phase
depicted in Figure 11 are needed.
REFERENCES
Ames BN, McCann J, Yamasaki E. 1975. Methods for detecting carcinogers and
mutagens with the Salmonella/mammalian microsome mutagenicity test. Mutat.
Res. 3L:34~-364.
Brusick DJ, Young RR. 1981. IERL-RTP procedures manual: Level 1
environmental assessment biological tests. EPA-60Q/8-81-02»*. Litton
Bionetics, Inc. Kensington, MD. pp 138.
Comnittee on Chemical Safety. 1979. Safety in academic chemistry
laboratories. 3rd ed. Amer. Chem. Soc. Pub.
EPA. 1979. Environmental Protection Agency. Method 625 - base/neutrals,
acids and pesticides. Vol. 4a, No. 233. Fed. Reg. pp. 695^8.
Hughes TJ, Pellizzari E, Little L, Sparacino C, Kolber A. 1980. Ambient air
pollutants: collection, chemical characterization and mutagenicity tasting.
Mutat. Res. 76:61-83.
Lentzen DE, et al. 1978. IERL-RTP procedures manual: Level 1 environmental
assessment. 2nd ed. EPA-600/7-78-201 (NTIS PB 293795), Research Triangle
Institute, Research Triangle Park, NC. pp. 279.
National Institute for Occupational Safety and Health. 1977. Carcinogens -
working with carcinogens. Publication No. 77-206. Department of Health.
Education, and Welfare, Public Health Service.
Occupational Safety and Health Administration 1976. OSHA safety and health
standards, general industry. OSHA 2206, 29CFR1910.
Tabor MV, Loper JC. 1980. Separation of mutagens from drinking water using
coupled bioassay/analytical fractionation. Int. J. Environ. Anal. Chem.
8:197-25.
D. S. National Cancer Institute. 1981. Office of Research Safety. The safe
handling of chemical carcinogens in the research laboratory. Presented at the
University of Cincinnati. Chicago, IL: IIT Research Institute.
Williams LR, Preston JE. 1982. Standard procedures for conducting the
Salmorella/microsomal mutagenicity assay. U, S. Environmental Protection
Agency. EMSL.
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APPENDIX B
ORIGINAL CONSENSUS PROTOCOL FOR DRINKING WATER
The original consensus protocol for preparing water samples, published as part
of the meeting proceedings (ICAIR ed., 1985) is attached in this report as Appendix
B for detail descriptions of the sample preparation procedures.
PROTOCOL FOR THE PREPARATION OF DRINKING WATER FOR MUTAGENICITY TESTING
1.0 Scope and Application
1.1 This method is used for the Isolation/preparation of residue organics
from drinking vacer for mutagenicity testing. Drinking water is defined as
water intended for human consumption.
1.2 This method is applicable to the isolation of residue organics from
drinking waters purified from both surface and ground sources. The method may
be applicable to other waters (e.g., see Wastewater Protocol).
1.3 The method provides for the reproducible qualitative recovery of muta-
genic residue organics absorbed by XAD resins from drinking water. Residue
organics are defined in Section 3.2.
1.4 This method does not provide information on volatile organics or on
highly polar and/or Ionic organics chat may be present In tfae water sample.
1.5 The apparatus employed In this method is portable, and its use is
straightforward. This allows for application to a wide variety of drinking
water sources In the field, and thus does not require the transport of water
samples to the laboratory.
1.6 This method is restricted to use by, or under the supervision of,
analysts experienced in chromatography and properly trained in the handling
and use of blohazardous materials.
2.0 Summary of Methods
2.1 A field duplicate sample of drinking water is passed through specially
designed chromatography columns containing polystyrenedivlnylbensene copoly-
mers and polymethacrylate polymer stationary phases. Following passage of the
sample through the columns, the residue organics are eluted from the collec-
tion system components with organic solvents, the solvents are removed by
evaporation and the remaining nonvolatile residue organics are stored under
nitrogen until mutagenicity testing is conducted.
2.2 The method described in this protocol is based on reports by LeBel et
al. (1979), Loper et al. (1983, 1984), Tabor and Loper (1984) Baird et al.
(1981), Jenkins et al. (1983) and Nellor et al. (1984) for the isolation of
residue organics from drinking water. For the purposes of this method, resi-
due organics are those which are absorbed by resins under the conditions
described herein and recovered by the solvent elutlon method of this protocol.
The procedure may not recover the highly polar and ionic organic species or
the highly volatile, low molecular weight organics. The design and testing of
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the large-volume, >50 L and small-volume, <50 L sampling apparatuses is given
in Loper et al. (1982, 1984), Tabor and Loper (1984) and Nellor et al.
(1984). These tests, for example, have shown that the large sampling
apparatus is capable of accommodating 1,100 L of low total organic carbon
water (i.e., drinking water), and that the small sampling apparatus Is
capable of accommodating more than 200 L of similar water, both without
apparent breakthrough of mutagenic residue organlcs. Additionally, the
passage of chlorinated, 2-ppm, ASTM Type I water through the system did not
produce residue organlcs that gave positive results in the Salmonella
mutagenicity test using strains TA98 and TA100 in the absence and presence of
metabolic activation. These studies established operation parameters such as
flow rates and line pressures. The results of these studies show that the
method provides reproducible qualitative recoveries of mutagenic residue
organlcs from a wide variety of drinking waters prepared from ground and
surface sources.
3.0 Definitions
3.1 ASTM Type I Water—
The American Society for Testing Materials (ASTM) defines Type I water
as having a maximum total matter of 0.1 mg/L, a maximum electrical conduc-
tivity at 25 C of 0.06 umho/cm, a minimum electrical resistivity at 25 C of
16.67 Mohm*cm and a minimum color retention time for potassium permanganate of
60 min.
3.2 Drinking Water—
Water Intended for human consumption.
3.3 Field Duplicate Samples—
Two samples taken at the same time and place, under identical circum-
stances, and treated exactly the same throughout field and laboratory pro-
cedures. Analysis of field duplicates indicates the precision associated with
sample collection, preservation and storage, as well as with laboratory
procedures.
3.4 Liter Equivalent—
The amount of residue organlcs concentrated from one liter of water.
3.5 Resin Blanks—
Two types of XAD resin blanks are required. The first, a chemical
contamination blank, is determined on an extract of the resin via gas chroma-
tography. The second, a mutagen contamination blank, is determined by muta-
genesis assay of residue organlcs eluted from the resins following passage of
ASTM Type I water through the assembled collection system.
3.6 Residue Organlcs—
Those organlcs adsorbed by XAD resins under the conditions described
herein and recovered by the solvent elutlon method of this protocol.
3.7 Solvent Blank—
Elutlon solvents are concentrated 1,000 times, and the concentrates
are bloassayed for mutagenesis.
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4.0 Interferences
4.1 The polystyrenedivinylbenzene copolymer stationary phase, XAD-2, and
the polymethacrylate polymer stationary phase, XAD-7, should be cleaned exten-
sively prior to use according to the methods described in Section 7.1 and
analyzed according to the methods in Section 9.2.
4.2 Interferences to the mutagenicity tests from elution solvents will
vary from supplier to supplier and from grade to grade. Therefore, it is
recommended that pesticide-grade or equivalent solvents are used and that
appropriate tests of each lot of solvent are conducted according to the pro-
cedures described in Section 9.1.
4.3 Interferences to mutagenicity tests and subsequent chemical analyses
from the apparatus can be avoided by using the stainless steel apparatus or
its equivalent, described in Section 6.1, allowing Che sample and solvents to
come into contact only with properly cleaned glassvare, described in Section
6.11, and utilizing only TFE tubing and TFE sealants where required.
5.0 Safety
5.1 The toxicity and carcinogenicity of the residue organic samples gener-
ated in this method have not been defined; however, each sample should be
treated as a potential health hazard. Procedures for handling such materials
have been described (USNCI 1981).
6.0 Apparatus and Equipment
6.1. Apparatus—
The general design of the sampling apparatus is shown in Figure 9, and
specifics of the design are shown in Figure 10.
6.1.1 Gauge, 0-100 psi, stainless steel with TFE diaphragm (Veriflo Model
IS-101S25DG or equivalent).
6.1.2 Flow control valve, one-way, needle, stainless steel (Whitey
SS-IRM4-S4 or equivalent).
6.1.3 Bacterial filter holder—
6.1.3.1 For high total organic carbon (20 ppm) or particulate-laden water, a
142-am sanitary filter holder, 316 stainless steel (Millipore YY30 142 36 or
equivalent) fitted with 1/4-in stainless steel pipe to tube male couplings
(Swagelok SS400-1-4-316 or equivalent).
6.1.3.2 For low total organic carbon (20 ppm) or low particulate-laden water,
a 47-om high-pressure filter holder, 316 stainless steel (Millipore XX45 047
00 or equivalent) fitted with l/4-in stainless steel pipe to tube male cou-
plings (Swagelok SS4DO-I-4-3L6 or equivalent).
6.1.4 Bacterial filters—
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Organic Residue Collection Unit
Regulators
Flow Pressure
Inlet
Glass Fiber ^
+/-3.Op Fluoropore
0.45U Durapore
I
Silanized Glass Wool Column
AOvi Frits
Filter
Unit
XAD-2 Column
AOvi Frits
XAD-7 Column
Outlet
tire 9. Schematic of nonvolatile residue organics concentration apparatus.
-------
Sample Chamber
Cauge 0-100 psl
Whltey
SS-1RM4-S4 ,
cH
Nylon
Male
Garden
Hose
1/4 in MPT
m
200 cc Each
Ver1f lo
Model
IR40IS250G
Bn
in
26 1/2 in
Exploded View
40 Micron Sintered
Stainless Frit
n
i
«sJJ
a o
1/4 in Swagelofc
I in Swagelok
re 10. Details of nonvolatile residue organics concentration apparatus.
(Note: prefill rat ion units are not shown.)
-------
6.1.4.1 Mlcrofilter glass discs without binder resin (Mllllpore Type AP40,
AP40 047 05) for lov total organic carbon filter holder.
6.1.4.2 Hydrophilic 0.45-oicron Durapore filter (Mllllpore NVLP D4700 or
equivalent) for lov total organic carbon filter holder.
6.1.4.3 Hydrophilic 0.45-micron Durapore filter (Mllllpore HVLP 142 5D or
equivalent) for high total organic carbon filter holder.
6.1.5 Connecting tubing, 316 stainless steel fitted with 1/4-in female
Svagelok stainless steel fittings (Svagelok SS402-1-316, SS403-1-316,
SS404-1-316 or equivalent). Any source of 316 stainless steel 1/4-in tubing
acceptable.
6.1.6 Sesin and glass vool columns—
6.1.6.1 Large sample volume (>50 L) columns of 200-cc bed volumes are
constructed, as shovn in Figure 10, of 1-in by 26.5-in 316 stainless steel
tubing fitted vlth 1-in female Svagelok stainless steel fittings (Svagelok
SS1012-1-316, SS1613—1—316» SS1614-1-316 or equivalent) and a 1-in male
Svagelok stainless steel cap (Svagelok SS1610-C-316 or equivalent) modified as
follovs. The center of the 1-in cap is tapped and threaded to accept a 1/4-in
pipe male coupling. The cap is fitted vlth a 1/4-in stainless steel pipe to
tube male coupling (Svagelok SS400-1-4-316 or equivalent) vhich is silver-
soldered in place. Prior to installation, the 1/4-ln coupling is machined
internally from the tubing side for an opening 3/16-in vide and 3/4-in deep.
This opening is then fitted vith a 5/32-in diameter by 1/8-in 40-micron
sintered 316 stainless steel frit. Both column ends are fitted vlth the
40-mlcron sintered 316 stainless steel frits. This entire column assembly is
available from Tristate Controls, Inc., 4303 Kellogg Avenue, Cincinnati, OH
45226.
6.1.6.2 Small sample volume (<50 L) columns of 25-cc bed volumes are con-
structed as shovn in Figure 2 and described above, except that the columns are
constructed of 1/2-in by 13-in 316 stainless steel tubing fitted vlth 1/2-in
female Svagelok stainless steel fittings (Svagelok SS812—1—316, SSS13—1—316.
SS814—1—316 or equivalent), the 40-oicron sintered 316 stainless steel frits
and 1/2-in male Svagelok stainless steel caps (Svagelok SS810-C-316) modified
as described above. This entire column system is available from Tristate
Controls, Inc., 4303 Kellogg Avenue, Cincinnati, Oti 45226.
6.2 Miscellaneous Apparatus Components—
(vhen line pressure of drinking vater exceeds supplies 40 psi).
6.2.1 Pumping—The lov line pressure drinking vater sample source may be
pumped through the columns by connecting a control volume TFE diaphragm pump
(Milton Roy Co., model NR-117S or equivalent) betveen the drinking vater
outlet tap and the collection apparatus (Section 6.1.1). The pump should be
capable of delivering 2.5 U.S. gal/h and developing a test pressure of
1,000 psi. The pump is fitted vlth a flow control valve, described in Section
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6.1.2, to regulate flow rates. The pump Is usually used when large sample
volumes (>60 L) are to be collected.
6.2.2 Positive displacement—The low line pressure drinking water samples
are collected in 20-L stainless steel reservoirs (Amicon Corporation model
8S20 stainless steel reservoir or equivalent). The apparatus (Section 6.1.)
is connected to the outlet of the reservoir by stainless steel tubing/
fittings. The inlet is connected to a nitrogen or helium gas cylinder fitted
with a pressure regulator. The reservoir system is usually used when small
volumes (<60 L) are to be collected.
6.3 Sample Storage Vials (Wheaton "500" amber serum battles Nos. 223778,
223779, 223785 or 223787 or equivalent), sealable with Teflon-faced septa
(Wheaton No. 224167 or 224172 or equivalent).
6.<« Resin Eluate Solvent Evaporator for Initial reduction of volume
(Buchi/Brinkman Rotary Evaporator, Model IS 00 500-9 or equivalent).
6.5 Sample Evaporator (Organomation Associates, Inc.) N-Evaps Model 111 or
equivalent).
6.6 Analytical Balance - Readable to 0.01 mg with a precision of t 0.01 mg.
6.7 Evaporation, displacement and sample storage gas, nitrogen or helium,
water compressed, high-purity grade.
6.8 Special Glassware—
6.8.1 Evaporative concentrator, modified micro Snyder, 4-oL tube (Konces No.
K-569250 or equivalent).
6.8.2 Soxhlet extraction apparatus, Corning series 3840 or equivalent.
6.9 Muffle Furnace capable of sustaining 500 C, for use in glassware
decontamination.
6.10 Solvent Reservoir 4-L stainless steel (Amicon Corporation, Model RS4
stainless steel reservoir or equivalent).
6.11 Flame Ionization Gas Chromatographic Unit fitted with a 6-ft by 4-ma
glass column containing 10Z 0V-101 (or equivalent) on 100/120 mesh GAS-CHROM-Q.
This unit oust be capable of temperature programming.
6.12 Cleaning of Apparatus and Glassware—
6.12.L Apparatus—Detergent wash, rinse with Cap and ASTM Type I water,
fallowed by successive rinses with pesticide-grade acetone and hexane.
6.12.2 Forty-micron sintered 316 stainless steel frits. Clean by sonicatlon
for 15 min in 6N nitric acid, followed by three successive 15-mln sonicatlons
in ASTM Type I water. The frlt3 are rinsed successively with pesticide-grade
acetone and hexane.
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6.12.3 Sample vials—Detergent wash, rinse with tap and ASTM Type I water,
followed by drying/muffling overnight at 500 C. Tightly wrap vials in
aluminum foil for storage until use.
6.12.4 Septum—Clean as for apparatus, Section 6.12.1.
6.12.5 General glassware—Clean as recommended in 44 TO 69464, December 3,
1979.
7.0 Reagents and Consumable Materials
7.1 Stationary Phases for Concentrating Nonvolatile Residue Organics—
Polystyrenedivinylbenzene copolymer, XAD-2, resin (Rohm and Haas Co.)
and polymethacrylate polymer, XAD-7, resin (Rohm and Haas Co.) are available
from numerous distributors. The XAD resins, as supplied, are contaminated
with extractable monomeric and polymeric species that must be removed before
use. The XAD-2 and XAD-7 resins are cleaned individually, but.using the same
procedure. The resin clean-up, detailed below, Involves removal of fines,
followed by Soxhlet extraction using a series of organic solvents. The
purified resins are stored as an acetone slurry in amber bottles until use.
Alternatively, the SAD resins, purified according to recommended methods and
specifications, are available with certification of analysis from the Munhall
Company, 5850 High Street, Worthington, OH 43085.
7.1.1 Resin cleaning—The resins can be cleaned in batches of 500 g, enough
for one sampling plus required blanks, or multiples thereof. The following
procedure is for 500-g batches, but can be scaled up as required.
7.1.1.1 To wash the resins and remove fines, transfer 500 g of the resin to a
1-L beaker and fill with ASTM Type I water. Slurry and allow to settle.
Decant the supernatant fluid containing the fines and repeat until supernatant
fluid is clear. This process may have to be repeated as many as ten times to
obtain a clear supernatant fluid.
7.1.1.2 Following removal of the fines, the moist resin is transferred to a
glass extraction thimble fitted with a fritted disc, and then the thimble Is
inserted into the Soxhlet extraction apparatus. For each 500 g of resin, the
following extraction sequence is conducted using 1 L of solvent for each
extraction. First the resin is extracted with 1 L of methanol for 8 h,
followed by a 14-h extraction with an additional 1 L of fresh methanol. The
22-h methanol extraction is followed by extraction with two 1-L portions of
methylene chloride, 8 h and 14 h, respectively. The 22-h methylene chloride
extraction is followed by extraction with two 1-L portions of hexane, 8 h and
14 h, respectively. Finally, the resin is extracted with two 1-L portions of
acetone, 8 h and 14 h, respectively. The resin is rinsed from the thimble
Into an amber bottle using a fresh portion of acetone. At this point, 20 cc
of resin is taken for resin blank analysis, as described in Section 9.2 (see
USEPA 1978). The resin is stored under acetone.
7.2 Glass Wool—
Silanlzed (Supelco Mo. 2-0411 or equivalent)
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7.3 Solvents—
Methanol, methylene chloride, hexane and acetone, pesticide-grade or
equivalent, and dimethylsulfoxide, DMSO, reagent-grade or equivalent, stored
in original containers and used as received.
7.4 ASTM Type I water, generated by a Continental/Millipore Water
Conditioning system (Tabor and Loper 1980) or equivalent.
7.5. Celite 545—
Prevashed as follows: Slurry 75 g of the filter aid in 500 mL ASTM
Type I water by swirling, then settle briefly and decant the supernatant fluid
containing the fine particles; repeat the process with a second aliquot of
ASTM Type I water. Pack a 25-cc stainless steel column, fitted at the outlet
end with a frit, with a 2-g plug of glass wool, then fill partly with ASTM
Type I water. Slurry pack the column with the Celite 545, using Type I water
as a liquid vehicle. Fit the inlet end of the column with a stainless steel
frit, followed by the Swagelok fittings. Connect Che Inlet end of the column
to a 4-L stainless steel reservoir (Amlcon Corporation, model RS4 or equiva-
lent) . Successively wash the column with one liter each of pestlclde-grade
acetone, hexane and acetone, according to the procedure in Section 6.12.2.
Following the last acetone wash, cap the column with 1/4-ln stainless steel
Swagelok plugs for storage of the column until use (within one week).
8.0 Residue Oryanlcs Isolation Procedure
8.1 Packing Procedure for 200-cc and 25-cc Columns—
The XAD-2 resin and XAD-7 resin columns are slurry packed, using
acetone as a liquid vehicle. After packing, label flow direction and seal
columns with 1/4-ln stainless steel Swagelok plugs (Swagelok ss-400-P-316) for
storage at 4 C until use (within one week). Do not allow the resins to dry;
keep them covered with solvent. The glass wool preflltratlon column is firmly
but not tightly dry packed with 25 g of silanized glass wool, using a clean
glass rod to position the packing material. Note: Che stainless steel frits
are omitted from the inlet end of the glass wool column. Label Che flow
direction of the column.
8.2 Apparatus for Drinking Water Sources with Line Pressure >40 psi—
The apparatus is assembled with proper flow directions for the
columns, as shown in Figure 9. and is connected to the sample source via an
appropriate fitting to the pressure regulator. When the Celite 545 column is
required, e.g., for wastewaters, insert this column in-line between the glass
wool column and the bacterial filter. When all of the filters and columns are
connected in-line, turn on the water and partially open the valve to gently
displace the acetone. Following the passage of one system volume of water
through the apparatus, open the valve fully and adjust the flow rate. If
using the large columns, sec Che regulator to 30 to 35 psi. For use of the
large columns, adjust the flow rate Co no greater than 250 mL/aln by setting
the pressure regulator to no greater than 30 psi (Tabor and Loper 1984, Loper
ec al. 1984). For use of the small columns, adjust the flow rate to no
greater than 100 mL/mln.
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8.3 Apparatus for Drinking Water Sources at Line Pressures <40 psl—
The apparatus Is assembled with proper flow directions for the
columns, as shovn in Figure 9.
8.3.1 Displacement of drinking water through the collection apparatus using
nitrogen or helium—Connect the gas line to the inlet of a 20-L stainless
steel reservoir containing the sample. Open the gas flow partially to gently
displace the acetone. Following the passage of one system volume of water
through the apparatus, further open the gas flow and adjust the tank pressure
regulator to 30 psl. Flow rates can be regulated via the in-line valve. If
the water sample is larger than 20 L or If the filters need to be changed, the
flow nay be stopped by closing the nitrogen tank and releasing the pressure
via the pressure release valve on the reservoir. Following the required
operation, changing of filters or connection to a new sample reservoir, close,
the pressure release valve and open the gas tank to resume flow; continue
until all the sample has been displaced over the system.
8.3.2 Use of the in-line pump—The drinking water supply is connected to the
pump inlet with appropriate fittings. This connecting line must be filled
with water before the pump Is turned on. The apparatus is connected to the
pump, and the drinking water sample is pumped through the system. Note that a
gentle flow of water is required in the beginning (Section 6.2) to displace
the acetone.
8.4 Bacterial Filters—
When the flow rate decreases to approximately 50% of the initial rate,
the bacterial filters probably need to be replaced. If the majority of the
sample, >90Z, has been concentrated, continue collection. If not, then
discontinue the concentration operation, disassemble the bacterial filter
holder and replace the filter(s) with fresh ones. The used filters are placed
in amber bottles for storage until extraction. Handle these filters with
forceps, since they are contaminated with bacteria, etc.
8.5 Volume Measurement—
The flow from the apparatus Is collected in an appropriate measuring
container. Usually 55-gal drums or smaller containers are used. At the end
of the collection process, the total collected volume is recorded.
8.6 At the completion of a collection, the apparatus is disassembled in
che order from the XAD-7 column, the last component in the system, to the
drinking water source. All columns are sealed with stainless steel Swagelok
plugs (Swagelok ss-400-P-316). Bacterial filters are removed from the holder
and stored in amber bottles until extraction. Water in che glass wool column
is allowed to drain, and the column is plugged as with the XAD columns.
Columns and filters are stored at 4 C until extraction, usually within 3 days.
The system should not be subjected to extremes of pressure or temperature.
8.7 Extraction of Residue Organics—
Resin columns and other system components, i.e., glass wool and
filters, are extracted with a hexane:acetone solvent system, 85:15 by volume,
according to the methods of LeBel et al. (1979), Loper ec al. (1983, 1984) and
Tabor and Loper (1984).
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9.7.1 XAD columns—The XAD columns are individually mounted in an upright
position so chac Che effluent end of the column is at the top. The end plugs
on the cop of che columns are removed, and the column is immediately connected
via che cop fitting to a pressure reservoir containing four to eight column
volumes of hexane:acetone, 85:15 by volume, solvent. Following removal of the
caps from the bottom of the column (i.e., the original inlet end), a length,
6 Co 10 in, of 1/4-in stainless steel tubing Is connected immediately to the
bottom, and the free end of this tube is inserted into a cleaned glass separa-
tes ry funnel to collect the water in the column. Pressure is gently applied
(1 to 3 psl) to the system, and the column is allowed to fill with solvent.
After the solvent has filled the column, stop che flov for equilibration for
15 to ZO min. The eluate tube is removed from the separatory funnel and
inserted into a cleaned glass receiver. The system is pressurized for a flov
rate not greater than one column volume per 20 min. The aqueous sample in the
separacory funnel is extracted three times with equal volumes of pesticide-
grade methylene chloride. These extracts are added to the solvent eluates
from the column. The solvent eluates from the column are collected, passed
through a small column (1 cm by 3 cm for the 25-cc system and 1 cm by 10 cm
for the 200-cc system) of anhydrous sodium sulfate, and the eluates are
collected in a cleaned round-bottom flask for initial concentration via rotary
evaporation.
8.7.2 Glass wool columns—The columns are mounted upright and connected to a
solvent reservoir containing four column volumes of hexane:acetone, 85:15 by
volume, solvent. The top of the column Is fitted to a tube in a manner
similar to che XAD columns. The column is filled with solvent, which is
allowed to equilibrate for 20 min. Following the equilibration, the solvent
is replaced with fresh solvent, and the process is repeated. The extracts are
combined for concentration via rotary evaporation. The glass wool is removed
for further extraction. This is accomplished by Soxhlet extraction for 10 h
using a ratio of five volumes of the hexane:acetone solvent for each cc volume
of glass wool. The Soxhlet extracts are dried as before (Section 8.7.1) and
are reduced in volume by rotary evaporation.
8.7.3 Bacterial filters—Each filter change group is extracted with 100 ml
of the hexane:acetone solvent by soaking the filter in the solvent contained
in a beaker for 20 min. This process is repeated two more times. The com-
bined extracts are reduced in volume by rotary evaporation.
8.3 Concentration of Reduced Volume Extracts—
Following rotary evaporation, the volume of each extract is measured
and recorded. The volume of each extract Is reduced further using the micro-
Snyder apparatus. Usually a measured aliquot of an extract is concentrated at
one time, rather than concentrating the whole extract. The sample is gencly
heated using Che N-Evap bath. As the solution is concentrated, some
constituents may come out of solution. If so, a small volume of acetone may
be added to keep the components in solution until sufficient evaporation of
the solvent has occurred to azeotrope Che remaining hexane from the sample.
(a) Registered trademark.
B-10S
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Usually three to four additions of acetone are required. Final volumes of the
acetone concentrates of the residue organic samples are recorded, and these
samples are stored in Teflon-capped amber vials at -20 C until mutagenicity
testing. J At the time of bloassay, an aliquot of the residue solution is
removed from the sample vial; typically, this aliquot is adjusted to necessary
bloassay volume vlth DMSO. If it is necessary to know the mass of residue
organics per dose, the amount of organlcs should be determined gravimetrically
on a separate aliquot of the acetone solution of residue organlcs.
9.0 Quality Control
9.1 Solvent Blanks—
Samples of each lot of the hexane and acetone elutlon solvents and the
methylene chloride extraction solvent are concentrated for mutagenesis test-
ing. Tvo liters of each solvent and 2 L of the 85:15 hexane:acetone solvent
are reduced In volume via rotary evaporation to 20 mL. Each sample is further-
concentrated to 0.2 oL via the micro-Snyder evaporative concentrator. The
residue concentrate is mixed with 400 uL of DMSO and submitted for muta-
genicity testing, tvo doses in duplicate, vlth Salmonella tester strains TA98
and TA100, in the absence and presence of metabolic activation (Loper et al.
1982, 1984). If the mutagenic response for any of the bloassay tests is equal
to or greater than double the spontaneous rate, the solvent lot is rejected.
9.2 Resin Blanks—
9.2.1 Chemical contamination—The general procedure (USEPA 1978) for
residual extractable organlcs is followed to determine contamination of the
cleaned SAD resins. For each resin, a 20-g sample of resin is extracted for
22 h with 200 mL of acetone using a Soxhlet extractor. The 200-oL extracts
are reduced in volume to 10 mL via evaporation under nitrogen, as described in
Section 8.8. The concentrated extracts are analyzed by gas chromatography
according to the USEPA total chromatographable organlcs analysis procedure
(USEPA 1978). In this procedure, 5 uL of the extract are injected into a
flame ionization gas chromatographic unit fitted vlth a 6-ft by 4-mm glass
column containing 10Z OV-101 (or equivalent) on 100/120 mesh GAS-CHROM Qm.
Gas chromatography conditions: injector temperature 300 C, Initial oven
temperature 50 C, final oven temperature 250 C, temperature program rate 20
C/min, nitrogen carrier gas flowing at 40 mL/min, sensitivity 8 x, recorder
range 1 mV. Resins are recleaned if the chromatograms of resin extracts show
peaks greater than 10Z full-scale eluting 5 min or later after injection.
The small sample volume apparatus (Section 6.1.6.2) is assembled as
described. Tvo liters of the 85:15 hexane:acetone are eluted through the
system and concentrated. Repeat this process until a gas chromatographic run
of background is constant £10Z. Pass 1 L acetone through the columns,
followed by 40 L ASTM Type I water. Elute columns per Section 8.7, and
bloassay this residue. Mutagenic response of 1,000:1 concentrates should be
less than 2x spontaneous reversion rate in the assay.
In cases where the organic residue is to be used for chemical
analysis, it is desirable to characterize the solvent and water blank elutions
by GC/MS or other specific analyses. In these cases, it is also recommended
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chac chlorinated waters be dechlorinated with ferrous citrate (Cheh et al.
L979) prior to concentration in order to prevent resin artifacts from
interfering with chemical analyses.
10.0 Sample Storage
10.1 Acetone concentrates of the residue organics are stored in Teflon-
capped amber vials containing an inert gas (nitrogen or helium) atmosphere at
-20 C until mutagenicity testing or further chemical analysis. The initial
mutagenicity testing should be conducted within two weeks, since it has been
noted that the bioactivity of some residue organics decreases with time (Loper
and Tabor, unpublished).
10.2 If the initial bioassay of the residue organics results in toxicity or
a no-dose response, a second portion (50- to 100-L equivalents) of residue
organics Is separated via HPLC, and the collected fractions of eluates are
bioassayed for mutagenicity. The HPLC methodology is detailed in the protocol
attendant to this overall document on the preparation of residue organics.
11.0 Data Records
11.1 Records to be maintained include a general description of the
practices used in water treatment at the purification plant supplying the
drinking water being sampled; time and location of sample collection; opera-
tion parameters for sample concentration, including flow rates, pressures,
filter changes and volumes; details of resin preparation; data on quality
control of resins and solvents; volumes of concentrated extracts and storage
data and any unusual occurrences during the collection operation. Chain-of-
custody forms should be executed for each sample from the time of preparation
of the columns and of concentration of the water residue organics through the
mutagenesis testing.
11.2 These records will be maintained for the glass wool extracts, the bac-
terial filter extracts, each XAD resin extract, the companion blank water
system sample and each lot of solvents used in the extraction of a sample
set.
12.0 Calculations
12.1 From data records, the final volume and/or weight of each concentrate
is related directly back to the number of liters of drinking water concen-
trated. Mutagenesis data will be reported in terms of number of revertants or
mutations per liter equivalent of water. Therefore, it is imperative that
accurate records of all volumes be maintained throughout all operations.
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REFERENCES
ACS. 1980. American Chemical Society. Division of Environmental Chemistry.
Extended abstracts of papers - 179th National meeting Vol. 20(1), Washington,
DC: American Chemical Society.
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B-108
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B-115
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APPENDIX C
SAMPLE COLLECTION INFORMATION CENTER
For the purpose of chain-of-custody and sample collection information, a
sample collection record form was filled out by the field manager at the time of
collection. The record included pertinent information relating to sample collection
procedures, sample type and appearance, any treatments performed, and storage and
shipment methods. All of the completed forms obtained in this project are attached
to this report as Appendix C.
C-116
-------
SAMPLE COLLECTION RECORD
I. SAMPLE INFORMATION
NBS Reference Sludqe-Toxicity Characteristics Leachate Procedure (TCLP)
1. Sample NoXxtract Leachate, AIHL-B5-0401 Collection Date 7/9/85 & 9/24/85
(a)
2. * Sampling Site &tht.
3. Field Sampling Manager (on-site) Yi Y. Wang, aihl
4. Contractor AIHL-CPHF Contract No. CR810022-02-0
5. EPA Project Officer L.R. Williams Program Name Quality Assurance
6. Source Sampled 45f-HfSfl) Sproiide§C§y E?R*n8i?Ka£! I lES*"'1
7. Discharge Rate of Source (Volume/Time) N/A
8. Quantity Sampled/Units 3 to 6 L of TCLP leachate. freshly prepared for each iiquid-
liquia HXtldCLlun muLUud n'^aluuiian eiipegimontr
9. Sample description (liquid, slurry, solid extract, appearance, etc.)
odorless transparent aqueous liquid without visible suspended solids,
ne 3e.oiuie.ii'.a
10. Other Information as Applicable
Collection Temp. room temperature Sampling Location AIHL
pH adjust to 5.0 Sampling Technique TCLP extraction
Other none
II. HANDLING h SHIPPING
1. Describe Sample Treatment Prior to Shipping (e.g., transfers, extractrants,
stored undiluted, grinding, solvents used)TCLP extraction: sodium acetate buffer
extraction with shaking, centnfugation. filtration; prepared at AIHL, no transportation
2. ^le^^torage and Shipping Conditions
Container Temperature Light
X Amber Glass X Ambient X Shield from light
Polyethylene Bottle Refrigerate(0*to 4 *C)
Coated Bag or Bottle Freeze (-20*C)
Teflon or Tedlar Bags Dry Ice
Other
3. Approximate Time in Storage and Time in Shipping none
4. Sample shipped to N/A
5. Mode and Carrier for Shipping N/A
6. Comments The EPA/NBS sludge sample was shipped by Federal Express from EPA, Las Vegas,
to AIHL. Once received, it was stored in a refrigerator (0 to 4"C).
(This form should be completed by the on-site sampling manager and accompany each
sample.)
(a) Graphically illustrate site of collection.
N/A
C-117
-------
SAMPLE COLLECTION RECORD
I. SAMPLE INFORMATION
Stringfellow contaminated groundwater from an on-site well, OW-2 ,
1. Sample No. atut Collection Date '7/9/65
2. Sampling Site Stringfellow Hazardous Waste Site
3. Field Sampling Manager (on-site) Wilson Horn, 5AIC
4. Contractor SAIC/CDHS-TSCD Contract no . collaboration
5. EPA Project Officer L.R. Williams Program NamsQuality Assurance
6. Source Sampled on-site extraction well, OW-2
7. Discharge Rate of Source (Volume/Time) N/A
8. Quantity Sampled/Units collected in 10 1-gallon bottles
9. Sample description (liquid, slurry, solid extract, appearance, etc.)
clear,light brown ,aaueous liquid without visible suspended solids, no sediments,
foamed wBen poured * _
10. Other Information as Applicable
Collection Temp. ambient Sampling Location OW-2
pH 3.2-3.5 Sampling Techniquespigot on the well-
haad
Other slight sulfurous odor, collected after purge
II. HANDLING & SHIPPING.
1. Describe Sample Treatment Prior to Shipping (e.g., transfers, extractr^nts,
stored undiluted, grinding, solvents used) shipped in the original collection
containers at ambient temperature, stored at 4°C at AIHL-CDHS
2. Field Storage and Shipping Conditions
Container Tamperature Light
X Amber Glass X Ambient(collection & shipping)ihield from light
Polyethylene Bottle X Refrigerate(0*to 4*C)(storage at CDHS)
Coated Bag or Bottle Freeze (-20*C)
Teflon or Tedlar Bags Dry Ice
—— storage approximately 4 hr. before
3. Approximate Time in Storage and Time in Shipping^,^,^. approximately 36 hr. shipping
4. Sample shipped to AIHL-CDHS, 2151 Berkeley Way, Berkeley, CA 94704
5. Mode and Carrier for Shipping Federal Express- Aircraft with air freight coolers
6. Comments 5 1-gallon bottles of ASTM Type I distilled water were provided by SAIC as
fta-field and travel blank, and processed at AIHL later as the method blank.
(This form should be completed by the on-site sampling manager and accompany each
samale.)
(a) Graphically illustrate site of collection.
C-118
-------
SAMPLE COLLECTION RECORD
I. SAMPLE INFORMATION
1. Sample No Stringfellow contaminated groundwater well. UGB-8,
hlKL-lgfQ*2k 9/27/BS
2. " Sampling Site Strincfellow Hazardous Waste Site
3. Field Sampling Manager (on-site) Wilson Horn. SAIC
4. Contractor SAIC/CDHS-TSCD Contract No. collaboration
5. EPA Project Officer L.R. Williams Program Name ouaHr.v >ssnranep
6. Source Sampled upqradient well. UGB-B
7. Discharge Bate of Source (Volume/Time) 30 gallons per minute
8. Quantity Sampled/Units collected in 10 1-gallon containers
9. Sample description (liquid, slurry, solid extract, appearance, etc.)
clear colorless odorless aqueous liquid without visible suspended solids, no
bnd.uitum.fe, nu Cudiiuay wimn pourua
10. Other Information as Applicable
Collection Temp. ambient Sampling Location una-a
pH 7.1-7.3 Sampling Technique hosino with a pump
Other hosing directly after 4000 gallon purge
II. HANDLING i SHIPPING
1. Describe Sample Treatment Prior to Shipping (e.g., transfers, extractronts,
stored undiluted, grinding, solvents used)
original collection containers at ambient temperature, stored at 4°C at AIHL-CDHS
2. Field Storage and Shipping Conditions
Container Temperature Light
Amber Glass ^^®^^collection%& shippxoflJLX^^®^ from light
Polyethylene Bottle y Refrigerate(0'to 4 *C) (storage)
Coated Bag or Bottle Freeze (-20'C)
Teflon or Tedlar Bags Dry Ice
Other
~ storred for approximately 3 days
3. Approximate Time in Storage and Time in Shipping»ffiy-pr,n„ ,rprn,ln,,t,i,, ->
4. Sample shipped toAIHL-CDHS' 2151 Berkeley Way, Berkeley, "ca 94704
5. Mode and Carrier for Shipping hps with frp17hT
6. Comments SftTr lahorarnry HicMnprf »«!TM Typo T wafer uac canffi! « rh* fiolH trawl
and method blank.
(This form should be completed by the on-site sampling manager and accompany each
sample.)
(a) Graphically illustrate site of collection.
C-119
-------
SAMPLE COLLECTION RECORD
I. SAMPLE INFORMATION
Effluent from a municipal wastewater treatment plant,
1. Sample No. MtKl.9E g,lo; C&Iectfon Date 7/24/85
2. Sampling Site a local municipal wastewater treatment plant
3. Field Sampling Manager (on-site) Harold J. Singer, CWQCB
4. ContractorCalif. State Water Quality Control Boardcontract No. collaboration
5. EPA Project Officer L.R. Williams Program NamsQuality Assurance
6. Source Sampled municipal wastewater effluents
7. Discharge Rate of Source (Volume/Time) N/A
8. Quantity Sampled/units collected in 2 S-gallon containers
9. Sample description (liquid, slurry, solid extract, appearance, etc.)
clear aqueous liquid with light yellow tint, some fine particulate suspension,
10. 2&hird^nformation as Applicable
Collection Temp. ambient Sampling Locationon-site pipe/faucet
pH 6.2-6.5 Sampling Technique faucet
Other swampy sewage odor
11. HANDLING & SHIPPING
1. Describe Sample Treatment Prior to Shipping (e.g., transfers, extractrsnts,
stored undiluted, grinding, solvents used) shipped in the original collection
containers at ambient temperature, stored at 4 C at AIHL-CDHS
2. Field Storage and Shipping Conditions
Container Temperature Light
X Amber Glass X Ambientfcollection s shipping)Ihield from light
Polyethylene Bottle X Refrigerate(0*to 4*C)(storage at CDHS)
_____ Coated Bag or Bottle ______ Freeze (-20'C)
Teflon or Tedlar Bags _____ Dry Ice
Other
3. Approximate Time in Storage and Time in Shipping no storage, 10 min. shipping
4. Sample shipped to AIHL-CDHS, 2151 Berkeley way, Berkeley, CA 94704
5. Mode and Carrier for Shipping automobile
6. Comments 2 1-gallon ASTM Tvoe I distilled water, provided bv AIHL. was brought to the
site and returned to AIHL as the field, travel, and method blank.
I
(This form should be completed by the on-site sampling manager and .-iccompsny each
sample.)
(a) Graphically illustrate site of collection.
N/A
C-120
-------
SAMPLE COLLECTION RECORD
I. SAMPLE INFORMATION
Q
Brackish San Francisco Bay surface wate^ ,, . _ _ ^
1. Sample No. * Collection Date W85
2. Sampling Site Berkeley Marina pier
3. Field Sampling Manager (on-site) Yi Y. Wang, AIHL and K.J. OiBartolomeis. CPHF
4. Contractor AIHL-CPHF Contract No. CR810022-02-0
5. EPA Project Officer L.R. Williams Program name Quality Assurance
6. Source Sampled San Francisco Bay water
7. Discharge Rate of Source (Volume/Time) N/A
8. Quantity Sampled/Units collected in 12 1-qallon bottles
9. Sample description (liquid, slurry, solid extract, appearance, etc.)
clear salty augeous liquid without visible suspended solids, no debris, no sediments,
ne unusual odor
10. Other Information as Applicable
Collection Temp. ambient Sampling Locationend of the Pier'
—^—«——-——¦ rTOTTTTGTTi b ldt
pH 7.8-8.0 Sampling Technique grab
Other briny odor
II. HANDLING & SHIPPING
1. Describe Sample Treatment Prior to Shipping (e.g., transfers, extractrsnts,
stored undiluted, grinding, solvents used) no sanrolp treatment. g'nipnori in *-hp
original collection containers at ambient temperature, storage at 4 C at AIHL-CDHS
2. Field Storage and Shipping Conditions
Container Temperature Light
X Amber Glass x Ambient(collection & shipBinajiisllield from ll9ht
Polyethylene Bottle x Refrigerate(0*to 4'C) (storage)
_____ Coated Bag or Bottle Freeze (-20'C)
Teflon or Tedlar Bags Dry Ice
Other
3. Approximate Time in Storage and Time in Shipping no storage, 10 min. shipping
4. sample shipped to AIHL-CDHS. 2151 Berkeley Way. Barclay. r& S47n*
5. Mode and Carrier for Shipping aiit.nmnhi
6. Comments AIHL laboratory distilled ASTM Tvnp T w»r»r mt 3C tho rravel
and method blank.
(This form should be completed by the or.-site sampling manager and ccconiP?ny each
sample.)
(a) Graphically illustrate site of collection. Clty oi= soTWnnsate Bridge
SF
_^3ay jSyidge
Oakland / S.F.Bay
fil
er
C-121
Berkeley Marina
-------
SAMPLE COLLECTION RECORD
I. SAMPLE INFORMATION
Surface runoff from a class I hazardous waste laadfill,
1* SamPle 'AIIIL OP 0 too SotlecKon bate 9/1V35
2. Sampling Site an on-site pond
3. Field Sampling Manager (on-site) Harold J. Singer, CWQCB
4. Contractor Calif. State Water Quality Control Boarflontract No. collaboration
5. EPA Project Officer L.R. Williams Program Name Quality Assurance
6. Source Sampled runoff from a hazardous waste disposal facility
7. Discharge Rate of Source (Volume/Time) none
8. Quantity Sampled/Units • collected in 10 1-gallon containers
9. Sample description (liquid, slurry, solid extract, appearance, etc.)
clear light yellow odorless aqueous liquid without visible suspended solids, no
aedxmt.il l 3
10. Other Information as Applicable
Collection Temp. ambient Sampling Location on-site pond
pH 7.2-7 .6 Sampling Technique grab
Other none
11. HANDLING 5 SHIPPING
1. Describe Sample Treatment Prior to Shipping (e.g., transfers, extractrsnts,
stored undiluted, grinding, solvents used) no sample treatment, shipped in the
original collection containers at ambient temperature, storage at 4~C at AIHL-CDHS
2. Field Storage and Shipping Conditions
Container Tamper a tuce Light
X Amoer Glass X Ambient*collection Shipping) xShield from light
Polyethylene Bottle x Refrigerate(0*to 4*C) (storage)
_____ Coated Bag or Bottle Freeze (-20*C)
Teflon or Tedlar Bags Dry Ice
Other
3. Approximate Time in Storage and Time in Shipping no storage. 45 mm. shipping
4. Sample Shipped to AIHL-CDHS. 2151 Berkeley Way. Berkeley. CA 94704
5. Mode and Carrier for Shipping automobile
6. Comments AIHL laboratory distilled ASTM Type I water was served as the field,
travel, and method blank.
(This form should be completed by the on-site sampling manager and accompany each
sample.)
(a) Graphically illustrate site of collection.
N/A
C-122
-------
SAMPLE COLLECTION RECORD
I. SAMPLE INFORMATION
Brackish surface water at the discharge site of an industrial wastewater
I. Sample No. nc Collection Date n /n /as
LieuUueiiL plant.; iMHL 85 ¦ 044fb
2. Sampling Site effluent discharge site in the San Francisco Bay
3. Field Sampling Manager (on-site) Yi Y. Wang, AIHL
4. Contractor AIHL-CPHF Contract No. CR810022-02-0
5. EPA Project Officer L.R. Williams Program Name Quality Assurance
6. Source Sampled mix of the brackish bay water and industrial effluents
7. Discharge Rate of Source (Volume/Time) more than 1 million gallon per day
8. Quantity Sampled/tJrtits collected in 3 1-gallon bottles
9. Sample description (liquid, slurry, solid extract, appearance, etc.)
clear salty aqueous liquid without visible suspended solids, no sediments,
Uytaly uily ud UI
10. Other Information as Applicable
mixing point of
Collection Temp. ambient Sampling Location en-o
pH TjJ. Sampling Technique grab
Other briny, slightly oily odor
11. HANDLING & SHIPPING
1. Describe Sample Treatment Prior to Shipping (e.g., transfers, extractrants,
stored undiluted, grinding, solvents used) shipped in the original collection
containers at ambient temperature, storage at 4 C at AIHL-CDHS
2. Field Storage and Shipping Conditions
Container Tamp? rata re Light
Amber Glass x Ambientfcollection shipping) XShield from light
Polyethylene Bottle x Refrigerate(0'to 4*C) (storage at CDHS)
Coated Bag or Bottle Freeze (-20*C)
Teflon or Tedlar Bags Dry Ice
Other
, » _ _ . _ storage approximately 3 hr. before
3. Approximate Time in Storage and Time in Shipping '
aliiyping,—»0 win. ompping
4. Sample shipped to aihl-CDHS. 2151 Berkeley Wav, Berkeley, CA 94704
5. Mode and Carrier for Shipping automobile
6. Comments AIHL laboratory distilled ASTM Type I water was served as the field,
travel, and method blank.
(This form should be completed by the or.-site sampling manager and accompany each
sanrole.)
(a) Graphically illustrate site of collection.
N/A
C-123
-------
SAMPLE COLLECTION RECORD
I. SAMPLE INFORMATION
1. sample No. Effluent frotn an industrial wastewater 9/17/B5
AiHL^-jy^uuue —1"—'
2. Sealing Site a local industrial wastewater treatment plant
3. Field Sampling Manager (on-site) Harold J. Sinoer. CWQCB
4. Contractor CaUf" State Water SuaUty Boarfear>traet No. collaboration
5. EPA Project Officer L.R. Williams Program Name Quality Assurance
6. Source Sampled industrial wastewater effluents
7. Discharge Bate of Source (Volume/Time) 2 million gallons per dav
B. Quantity Sampled/Units collected in 10 1-qallon containers
9. Sample description (liquid, slurry, solid extract, appearance, etc.)
clear light yellow aqueous liquid without visible suspended solids, no sediments
10. Other Information as Applicable
Collection Temp. amhipnt Sampling Location on-site pipe
pH 7.1-7-5 Sampling Technique grab
other mnqrv with m/petroleum odor
II. HANDLING £ SHIPPING
1. Describe Sample Treatment Prior to Shipping (e.g., transfers, extractrants,
stored undiluted, grinding, solvents-used) nf) sample treatment, shipped in the
original collection containers at ambient temperature, storage at 4 C at AIHL-CDHS
2. Field Storage and Shipping Conditions
Container Temperature Light
v Amber Glass y Ambient (collection ft shipmno^hield from light
Polyethylene Bottle x Refrigerate(0*to 4*C) (storage)
Coated Bag or Bottle Freeze (-20*C)
Teflon or Tedlar Bags Dry Ice
Other
3. Approximate Time in Storage and Time in Shipping nn i hr. shinning
4. Sample shipped to AIHL-CDHS, 2151 Berkeley Way, Berkeley, CA 94704
5. Mode and Carrier for Shipping anmnnhiit
6. Comments ftTWT. lahnramry Hi ct l np* &STM Typo T waro- wag *e>
-------
APPENDIX D
PRIMARY AMES BIOASSAY DATA AND DOSE-RESPONSE
CURVES FOR THE PROTOCOL VALIDATION STUDY
The primary data of the Ames assay and graphs of the dose-response curves for
the protocol evaluation study using six generic types of environmental water and
wastewater samples are attached as Appendix D. The data are recorded on the
EPA-HERL (Health Effects Research Laboratory) In Vitro Result Form obtained from
Dr. Larry D. Claxton, USEPA, Research Triangle Park, North Carolina. An explanation
for the codes used on this form is listed in the following:
Research Lab ID: CPHF = California Public Health Foundation
AIHL 3 Air and Industrial Hygiene Laboratory
Experiment Date: Month, day, year of the date of the Ames assay performed
Test Sample Identification: See the Sample Identification Form for details
Activation Batch: CPHF-AIHL S-9 batch number
Test Type: 01 3 standard plate incorporation assay
Strain Batch Number: CPHF-AIHL bacterial strain batch number
Mammalian S-9: R 0 male Sprague-Dawley rats
L = liver
A = Aroclor 1254-induced
Remarks Made: 2 = no remarks
Phenotype Check Conclusion: 1 = true mutants
Technician/Personnel: KIC » Kuo-In Chang, the technical leader for the Ames assay
YYW =» Yi Y. Wang, the project manager
Solvent: 51 =¦ distilled water
54 = dimethylsulfoxide
Positive Controls: 01 = sodium azide
03 = 2-nitrofluorene
04 » 2-aminoanthracene
07 » 2-aminofluorene
10 o benzo(a)pyrene
11 = 4-nitrobenzoic acid
Background: blank = normal background lawn
2 <¦ partially clear, sparse lawn
3 = clear, no lawn
4 = contaminated
5 - precipitation and normal background lawn
6 ¦ precipitation and partially clear
7 » precipitation and clear
8 ¦ tiny pinpoint colonies with partially clear lawn
9 = tiny pinpoint colonies without background lawn
D-125
-------
Cfc frt O022—O2— 0 HERL IN VITRO SYSTEM
SAMPLE IDENTIFICA TION FORM (INTERIM)
SECTION >f. ENVIRONMENTAL WATERS AND MASTEWATErfL.
System ID
Sample ID
Lab Yt
No
Sample Description
A i\h L -\%\s\-
o\4\ol
Ia/35 *£ F^Ef^c\s\ slui>ge-\t\clA \l\e\a c\h a r|f | | | |
58 9 10
11-14
15-56
, , Sampfe 10
Lab Yt
No
Sample Description
AIUL-t$-\ovo 2
ST*1i\n$FE |c O^TAHx|^At|e1/> k h/A 7fl>e| o|m|2
5-8 910
11-14
15-56
t t Sampfe '0
Lab yf
No
Sample Description
0
STkl ZN CrFZLL DH Wfl|ErtTl tUlaM** IflkHffl
5-8 9to
11-14
15-56
Lab Sample ID
No
Sample Description
MxJoll -Mi- -
0*03
|m «|aJ i|c|j ?\a c| Iw\ast]ewATE^ Ip^UMtI \EFFLh\e»i\ I I I I I
58 9 10
11-14
15-56
Lab Sampye, 'D
No
Sample Description
3 A Aj| 1 f\sl Aficr
[5co bay blackish |s|M*/=A|ce wa|t-£«|
58 9 10
11-14
15 56
, . Sample ID
Lab *7
No
Sample Description
ArlrtlU" ** ~
»f+/4
B K|a|c|k isn Re c-bx v i\n$ j|w|p|m s tH i|a|l e f|f l «bMT 1 1 1
5-8 9-10
11-14
15-56
Lab SamPyer
No
Sample Description
a i n l
0 4-d 5
Stf|*|fAC£| RnhJ*FF\ F|fc|oM M |c|l|a|s5 -ll \l A\tf t> f i\l\l\ 1 1
55 9 10
11-14
15-56
1 Sample ID
r~i 1 J7 .
No
Sample Description
[Ajx w
0 f o\i]
r*i x> m s r er al
WASTEWATER \pLAhlT EpFi- V7 1
5 8 9-10
11 14
15 56
farms Com/jfetfon
Iwlwf 4
-------
TABLE 12. MUTAGENICITY OF AN EPA/NBS REFERENCE SLUDGE - TCLP LEACHATE,
SAMPLE NO. AIHL-85-Q401
A. Results of Initial Screening Experiments
2 3
Extraction Residue Sample Specific Mutagenic Activity (revertants/mg) '
<:xp. Date Method
Weight Volume
(mg) (L) -S9
TA98
2%S9 10%S9
TA100
-S9
2%S9 10%S9
7/9/85^ Base/Acid 5.3 3 887 1287
9/24/85 Base/Acid 12.1 6 990 1149
1071 674 600 812
918 < 460 < 483 < 502
B. Comparison of Mutagenic Response in TA98 with 2% S-9 Mix
Exp. Date
Residue
Weight
(mg)
Sample
Volume
(L)
Specific Mutagenic
Activity
(revertants/mg)
2,3
Mutagenic Response
per Unit Volume
(revertants/L)
7/9/85 5.3 3 1287 2274
9/24/85 12.1 6 1149 2308
1. The extraction method indicates the order of pH in liquid-liquid extraction.
2. The specific activity of the mutagenicity dose-response curve is represented by
the slope (b as shown in the statistical analysis on graphs in Appendix D).
3. The spontaneous mutation and positive control values are listed an the Strain
Function Tests, Cell Titer and Viability Record in Appendix F.
4. The optimum testing condition in the initial screening experiment was in TA98
with 2% S-9 mix.
5. Activity in the background control (sodium acetate butter) was observed; the
screening experiment was repeated after making corrective actions.
D-127
-------
TCLP-NBS WASTEWATER
DichloromGthane Extract
600
450 _
300 _
r
150 _
TA 98. -S9
SYMBOL"*
LINETYPE—
y=a+b*x
n-8
a=93.0471
b=8B7.0238
sM=67.5575
TA 98. 2% S9
SYMBOL^
LINETYPE=
y=a+b*x
n=8
a=56. 3834
b=1287. 1720
V=23.7862
TA 98. \DZ S9
SYMBOL-o
LINETYPE
y=a+b*x
n°8
an38.9795
b=1070.6829
s^B. 9735
s=28.6295
sk=171.1038
r=0.9042
soa10.0801
sk=60.2435
r=0.9935
9,»3.8028
sb=22.7274
r=0.9987
mg/plate 7/9/85
-------
HERL IN VITRO RESULTS FORM
~
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-------
HERL IN VITRO RESUL TS FORM
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-------
HERL IN VITRO RESUL TS FORM
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67 70
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-------
TCLP-NBS WASTEWATER
DichloromethanG Extract
600
450 _
150 <9
TA 100. -S9
SYMBOL"«
LINETYPE= —
ycc+b*x
n=8
a=191.5958
b=673.7580
s,.,=31.2027
TA 100. 21 S9
SYMBOL^
LINETYPE=
y=a+b*x
n=8
a= 192.7421
b-600.3570
s ,=10. 2341
TA 100, 102 S9
SYMBOL0o
LINETYPE
y=a+b*x
n=8
a=162.4421
b=812.2005
sm=12. 3922
s,= 13.2231
sk=79.0276
r=0. 9611
s0=4.3370
sb=25.9199
r=0. 9945
9,=5.2516
sk=31.3860
r=0.9956
mg/p1 ate
7/9/85
-------
HERL IN VITRO RESUL TS FORM
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66
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66
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066
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|^| S 9 Mm
©
67 70
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-------
TCLP-NBS WASTEWATER
DichloromQthane Extract
800
600 _
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TA 9B. -S9
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19 20
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66
Sterility
5 9 Mm
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25 30
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066
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I 6 7 70
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TABLE 10. MUTAGENICITY OF A CONTAMINATED GROUNDWATER FROM THE STRING-
FELLOW HAZARDOUS WASTE DISPOSAL FACILITY ON-SITE WELL OW-2,
SAMPLE NO. AIHL -85-0402
A. Results of the Initial Screening Experiment
2 3
Extraction Residue Sample Specific Mutagenic Activity (revertants/mg) '
Exp. late Method^ Weight Volume TA98 TA100
(mg) (L) -S9 2%S9 10%S9 -S9 2%S9 10%S9
7/22/85 Base/Acid 129 3 58 84 79
4
B. Comparison of Mutagenic Response in TA98 with 2% S-9 Mix
Extraction Residue 5ample Specific Mutagenic
Exp. Date Method^ Weight Volume Activity ^ 3
(mg) (L) (revertants/mg) '
<57 <61 <56
Mutagenic Response
per Unit Volume
(revertants/L)
7/22/85
Base/Acid
129
3
84
3598
8/2/85
Acid/Base
43
3
48
685
pH 2 fraction only
8/2/85
Acid/Base
7
3
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< 95
pH
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only
9/24/85
Acid/Base
60
4.5
< 53
< 702
10/17/85
Acid/Base
67
3
< 28
< 629
1. The extraction method indicates the order of pH in liquid-liquid extraction.
2. The specific activity of the mutagenicity dose-response curve is represented by
the slope (b as shown in the statistical analysis on graphs in Appendix D).
3. The spontaneous mutation and positive control values are listed on the Strain
Function Tests, Cell Titer, and Viability Record in Appendix F.
4. The optimum testing condition in the initial screening experiment was in TA98
with 2% S-9 mix.
D-164
-------
360
STRINGFELLOW WASTEWATER
DichloromethanG Extract(Sample 0402)
f
TA 98. -S9
SYMBOL= *
LINETYPE= —
y=a+b*x
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b=50.2372
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TA 98.21 S9
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a=43.6639
b=83.7664
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TA 98. 10X S9
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0=41.9181
b=79.1426
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sk=2. 2550
r=0.9825
s,=3. 8608
sh=3. 4324
r=0.9820
8.=2. 4864
sk=2.2105
r=0.9915
0. 0
mg/plate 7/22/85
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TABLE 11. MUTAGENICITY OF A CONTAMINATED GROUNDWATER FROM THE STRING-
FELLOW HAZARDOUS WASTE DISPOSAL FACILITY UPGRADIENT WELL UGB-8,
SAMPLE NO. AIHL-85-042A
Mutagenic Response in TA98 with 2% S-9 Mix
Extraction Residue Sample Specific Mutagenic Mutagenic Response
Exp. Date Method* Weight Volume Activity ^ per Unit Volume
(mg) (L) (revertants/mg) ' (revertants/L)
10/17/85 Acid/Base 3.8 3 13B 174
1. The extraction method indicates the order of pH in liquid-liquid extraction.
2. The specific activity of the mutagenicity dose-response curve is represented by
the slope (b as shown in the statistical analysis on graphs in Appendix D).
3. The spontaneous mutation and positive control values are listed on the Strain
Function Te3t3, Cell Titer and Viability Record in Appendix F.
4. The optimum testing condition for the Stringfellow OW-2 sample was in TA98
with 2% S-9 mix. The same condition was applied to the UGB-8 sample for
comparison purposes.
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120 _
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-------
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©66
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(£)
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Initials I I I jl
-------
TABLE 8. MUTAGENICITY OF A MUNICIPAL WASTEWATER TREATMENT PLANT
EFFLUENT, SAMPLE NO. AIH_-85-0403
A. Results of the Initial Screening Experiment
Exp. Date Method
2 3
Extraction Residue Sample Specific Mutagenic Activity (revertants/mg) '
Weight Volume
(mg) (L) -S9
TA98
2%S9 10%S9
TA100
-S9
2%S9 10%S9
8/2/85 Base/Acid
17
<33 <43
88
< 166 < 165 < 145
B. Comparison of Extraction Methods for Mutagenic Response in TA98 with 10% S-9 Mix
Extraction Residue Sample Specific Mutagenic
Exp. Date Method^ Weight Volume Activity _ -
(mg) (L) (revertants/mg) '
Mutagenic Response
per Unit Volume
(revertants/L)
8/2/85
8/13/85
10/17/05
Base/Acid
Base/Acid
Acid/Base
17 3
13 3
9 3
88
75
148
491
325
443
1. The extraction method indicates the order of pH in liquid-liquid extraction.
2. The specific activity of the mutagenicity dose response curve is represented by
the slope (b as shown in the statistical analysis on graphs in Appendix D).
3. The spontaneous mutation and positive control values are listed on the Strain
Function Tests, Cell Titer, and Viability Record in Appendix F.
4. The optimum testing condition in the initial screening experiment was in TA98
with 10% S-9 mix.
D-192
-------
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240
MUNICIPAL WASTEWATER
DichloromQthanG Extract
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25 30
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HERL IN VITRO RESUL TS FORM
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Conclusion
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@66
Sterility
| 1 1 S 9M»
(§)
67 70
k
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TABLE 6. MUTAGENICITY OF A BRACKISH SAN FRANCISCO BAY SURFACE WATER,
SAMPLE NO. AIHL-85-0404
A. Results of the Initial Screening Experiment
2 3
Extraction Residue Sample Specific Mutagenic Activity (revertants/mg) '
Exp. Date Method^ Weight Volume TA98 TA100
(mg) (L) -S9 2%S9 10%S9 -S9 2%S9 10%S9
9/24/85 Base/Acid 6.5 3 < 106 < 139 < 162 < 404 < 460 < 401
U
B. Comparison of Extraction Methods for Mutagenic Response in TA98 with 2% S-9 Mix
Extraction Residue Sample Specific Mutagenic Mutagenic Response
Exp. Date Method Weight Volume Activity ^ ^ per Unit Volume
(mg) (L) (revertants/mg) ' (revertants/L)
10/4/85
Acid/Base
4.8
3
177
283
12/17/85
Base/Acid
1.2
1.5
218
176
1. The extraction method indicates the order of pH in liquid-liquid extraction.
2. The specific activity of the mutagenicity dose response curve is represented by
the slope (b as shown in the statistical analysis on graphs in Appendix D).
3. The spontaneous mutation and positive control values are listed on the Strain
Function Tests, Cell Titer, and Viability Record in Appendix F.
4. Although results of all six conditions in the initial screening experiment were
below the detection limit, TA98 with 2% S-9 mix produced a questionable positive
response close to the detection limit. This condition was therefore used in the
follow-up experiments.
D-204
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s„=25.8682
r=0.9479
v < I;
56 0000
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4 1
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60
53
73
60
131
120
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0. 18
0. 27
0. 36
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12/17/85
-------
HERL IN VITRO RESUL TS FORM
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67 70
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11 m
-------
TABLE 7. MUTAGENICITY OF A BRACKISH RECEIVING SURFACE WASTEWATER FROM
THE DISCHARGE SITE OF AN INDUSTRIAL WASTEWATER TREATMENT PLANT,
SAMPLE NO. AIHL-85-044A
Comparison of Extraction Methods for Mutagenic Response in TA9B with 2% 5-9 Mix^
Extraction Residue Sample Specific Mutagenic Mutagenic Response
Exp. Date Method Weight Volume Activity ^ j Per Unit Volume
(mg) (L) (revertants/mg) ' (revertants/L)
11/20/85
Base/Acid
1.5
3
465
232
12/17/B5
Acid/Base
0.9
1.5
414
240
1. The extraction method indicates the order of pH in liquid-liquid extraction.
2. The specific activity of the mutagenicity dose response curve is represented by
the slope (b as shown in the statistical analysis on graphs in Appendix D).
3. The spontaneous mutation and positive control values are listed on the Strain
Function Tests, Cell Titer, and Viability Record in Appendix F.
4. The optimum testing condition for the San Francisco Bay water was in TA98
with 2% S-9 mix. The same condition was applied to the brackish receiving
wastewater for comparison purposes.
-------
BRACKISH WASTEWATER
Dichloromethane Extract
D
to
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360
270 _
180 _
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SYMB0L=a
LINETYPE
y=a+b*x
n=10
a=57.5335
b=464.5527
sh=\7.6846
x< i •>
0 0000
0 0000
0 0060
0100
0 1 00
1 000
1000
3000
3000
6000
s.=6. 9901
sk=29.5335
r=0. 9842
Y
66
50
56
40
44
1 20
1 14
217
214
314
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0. 00
0. 18
0. 36
0. 54
0. 72
mg/p1 ate
11/22/85
-------
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15 18
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25 30
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31 32
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33 38
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BRACKISH WASTEWATER
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100 _
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SYMBOL-a
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12/17/85
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-------
TABLE 5. MUTAGENICITY OF A SURFACE RUNOFF FROM A CLASS I LANDFILL, SAMPLE
NO. A[HL-85-0405
A. Results of the Initial Screening Experiment
i
2 3
Extraction Residue Sample Specific Mutagenic Activity (revertants/mg) '
Exp. Date Method^ Weiqht Volume TA98 TA100
(mg) (L) -S9 2%S9 10%S9 -S9 2%S9 10%S9
9/24/85 Base/Acid
15
122
88
86
< 242 < 230 < 200
B. Comparison of Mutagenic Response in TA98 without S-9
Exp. Date
Extraction
Method^
Residue Sample Specific Mutagenic Mutagenic Response
Weight Volume Activity _ ,
(mg) (L) (revertants/mg) '
per Unit Volume
(revertants/L)
9/24/85 Base/Acid 15 3 122 614
10/4/85 Acid/Base 31 3 <11 < 112
10/17/85 Acid/Base 11 3 < 325 < 114
1. The extraction method indicates the order of pH in liquid-liquid extraction.
2. The specific activity of the mutagenicity dose response curve is represented by
the slope (b as shown in the statistical analysis on graphs in Appendix O).
3. The spontaneous mutation and the positive control values are listed on the Strain
Function Tests, Cell Titer, and Viability Record in Appendix F.
4. The optimum testing condition in the initial screening experiment was in TA98
without S-9 mix.
5. No revertant colonies on all the testing plates, background lawn was normal
except the plates at the highest dose.
D-220
-------
SURFACE RUNOFF
DichloromQthanG Extract
140
105
70
35 _
o
o °
&
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* /
- * /
A
*
s** ;
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0. 00
J
TA 98. -S9
SYMBOL13 *
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y=a+b*x
n=13
o=30.4700
b=122. 4981
s,.,=9.9543
TA 98. 21 S9
SYMBOL=a
LINETYPE=—
y=a+b*x
n=13
0=44.9469
b=87.8681
=6. 9870
TA 98. 10Z S9
SYMBOL0o
LINETYPE
y=a+b*x
n=13
a°50.0619
b=85.8997
9M=8.6198
s0=3. 4369
sk=l 1.6508
r=0.9537
s=2.4124
s„=B. 1777
r=0.9555
s=2.9761
sk=10.0887
r=0.9318
0. 18
0. 36
0. 54
0. 72
mg/platQ 9/24/85
-------
HERL IN VITRO RESUL TS FORM
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31 32
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33 38
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D
66
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67 70
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19 20
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B
66
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TABLE 9. MUTAGENICITY OF AN INDUSTRIAL WASTEWATER TREATMENT PLANT
EFFLUENT, SAMPLE NO. AIHL-85-0406
A. Results of the Initial Screening Experiment
Extraction
Residue
Sample
2 3
Specific Mutagenic Activity (revertants/mg) '
Exp. Date
Method^
Weight
Volume
TA98
TA100
(mg)
(L)
S9 2%S9 10%S9
-S9 2%S9 10%S9
9/24/85
Acid/Base
48
3 <
35 <46 < 54
< 161 < 153 < 134
B. Results of Follow-up Experiments in TA98
with 10% S-9 Mix4
Extraction
Residue
Sample
Specific Mutagenic
Exp. Date
Method^
Weight
(mg)
Volume
(L)
Activity _ ,
(revertants/mg) '
10/4/85
Base/Acid
52
3
< 53
10/17/85
Base/Acid
62
1.5
< 38
10/17/85
Acid/Base
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1.5
< 38
C. Results
of Follow-up Experiments in TA9B
with 30% S-9 Mix5
Extraction
Residue
Sample
Specific Mutagenic
Mutagenic Response
Exp. Date
Method^
Weight
(mg)
Volume
(L)
Activity 2 3
(revertants/mg) '
per Unit Volume
(revertants/L)
2/11/86
Acid/Base
28
1.1
63
1585
2/28/86
Acid/Base
57
3
95
1810
1. The extraction method indicates the order of pH in liquid-liquid extraction.
2. The specific activity of the mutagenicity dose-response curve is represented by
the slope (b as shown in the statistical analysis on graphs in Appendix D).
3. The spontaneous mutation and positive control values are listed on the Strain
Function Tests, Cell Titer, and Viability Record in Appendix F.
4. Although results of all six conditions in the initial screening experiment were
below the detection limit, TA93 with 10% S-9 mix was the best condition in
which the result was closest to the detection limit. This condition was therefore
used in the follow-up experiments.
5. Modification of the S-9 condition (30% S-9 mix) was applied because of equivocal
results obtained in experiments with 10% S-9 mix.
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-------
APPENDIX E
PRIMARY AMES BIOASSAY DATA FOR THE RECOVERY STUDY
The worksheets, primary data, and dose-response curve graphs for the mutageni-
city recovery of three spiked reference mutagens: benzo(a)pyrene, 2-aminoanthracene,
and 4-nitrobenzoic acid, are attached as Appendix E.
WORKSHEET FOR THE SPRM RECOVERY STUDY IN THE AMES ASSAY
IN STRAIN TA98
Extraction Spiked Dose % Recovery
Method ( ug/1.5 L water ) 2% S-9 Mix 10* S-9 Mix
$ ***<./<£-£0 i
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RECOVERY EFFICIENCY
Benzo(a)pyrene Dose-Response
120 _
21 59
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RECOVERY EFFICIENCY
Benzo (a)pyrene DosG-RQsponsQ
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1 lolbU
1 1 4°l
Ik 4 J
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Mill
l/kl
~
11 \4?\s\
1 1 \i\o\
17/71
~
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~
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~
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~
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~
0
m
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~
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~
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Mill
~
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~
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~
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~
II 1 1
~
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~
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0
IJJ
~
MINI
LLIJJ
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~
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~
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~
IMII
~
0
m
~
111 II11
C D
Mill
~
1 1 1
~
II 1 1
M
Mill
~
Mill
~
0
m
~
II1 llll
IT1T1
11 1
~
1 1 1
~
II II
~
IMII
~
IMII
~
§
m
~
IIIUII
Mill
11 1II
~
Mill
~
II 1 1
~
Mill
~
IMII
~
0
111
~
IIIUII
Mill
II 1II
~
Mill
~
II 1 1
~
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~
IMII
~
0
LU
~
II1 llll
Mill
IMll
~
Mill
~
Mil
~
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~
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~
0
m
~
III Ul
Mill
IMII
~
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~
II 1 1
~
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~
0
m
~
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II 11
~
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D
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~
0
m
~
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~
Mil
~
II 1 1
n
llll
~
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~
0
m
~
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Mill
1 1
~
llll
~
II 1 1
11
llll
~
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~
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CD
~
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1 1 III
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LJ
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-------
ID
I
K>
Ol
VO
01
4->
o
Q.
\
(/)
4->
C
o
-p
L
Qi
>
QJ
en
CO
cn
480
RECOVERY EFFICIENCY
Benzo (a)pyrene DosG-RGsponsQ
360
240 _
120
/
/
9
/
/
O /
/
• /
/
/
/
/
Q
/
/
/
/
/
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y °
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/
/
/
/
/
/
/
*
~ °
r
/
'.
1
10X S9
SYMBOL-*
LIKETYPE--
y°a+b*x
n-17
aa28.9774
b=419«4168
25.7660
9
0.0
0.3
0.6
0.9
1.2
x< I >
0 0000
0 0000
0 0000
0500
0500
0500
1000
1 000
1000
5000
5000
.5000
7500
7500
7500
1 0000
1 0000
8048
sk=17.3171
r=0.9875
v
41
33
44
55
54
44
80
64
75.
284
213
195 .
306.
375
399
455
456
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0000
0000
0000
0009
0000
0000
0000
0000
PI0M0
0000
0000
0000
0000
0000
0000
0000
0000
ug/plato
12/17/85
-------
m
i
to
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CD
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c
~
-p
L
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Q)
(H
CD
CD
800
RECOVERY EFFICIENCY
500 ug B(a)p/1.5 1 Blank. BasQ/Acid
600 _
400 _
200 _
102 59
SYMBOL0*
LINETYPE—
y=a+b*x
n=18
a-44.3568
bc777.3025
sM=65.5862
a
0
X' i >
e 0000
0000
0000
0500
0500
0500
1000
1000
1000
5000
.5000
5000
7500
7500
7500
0000
0000
0000
9.-22.4543
9fc°40.7140
r=0.9787
Y <
51
50 .
46 .
78 .
53
48
99 .
104 .
31
493
455 .
434
726
715
748
750
674
780
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0000
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0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
ug/plate
12/17/85
-------
HERL IN VITRO RESUL TS FORM
~
i i
5 8
C F H Is
System ID
Research
Lab ID
9 14
/UlAihte
MO DA YR
Experiment Date
15 18 19 20 21 24
LAB YR NUMBER
Test Sample Identification
25 30
O
3
Activation Batch
t
31 32
Test Type
(table 101
33 38
39 42
1 l7WI;lirll7I?R?
f Strain Batch No
0 72 73 74 75
en cn
'ime Imml lemo IC
| Pre incubation |
Microorga nism
© 76 78
Animal
IL
Organ
L
Inducer
/I
/o % /-lrx
Remarks
Made*
Yes t
@65
QPhenocopy
Check
Conclusion
| Table I J|
I
66
Sterility
S 9 Mm
1 Not Contam
2 Contam
3 Not Checked
©
67 70
H
o
Activation
MutUte
Per Plate t$Jf
©_"
nsi
/S(
LLIc/
Sample
Sterility
Check
t Not Contam fime (mini Jemo lCentr4 Technician
2 Contam
3 Not Checked
0
Solvent
Positive
liable 111
43 44'
SOL
51
Dose Level
Q Units of
Concentration
Blank - mg/ml
2 -
45
~
o
Stock con-
centration
/Mr )
C/->}
\C""K '
®
Ami Per
Plate (til)
5/54
i
Plate A
©
Count
E
©
B
G
58 59 60 63
^~miQ
Plate B
©
Count
WW.
©
B
G
0
Plate C
©
Count
tst
3
m.
©
B
G
H
Plate D
©
Count
70 73
©
B
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7T
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Plate E
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Z£Z2
a
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Initials j j | |
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HERL IN VURO RESUL TS C ON J IN UA TION FORM
w
i
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I/IjI/I?I JBI
E
ft
15 18 19 20 21 24
UM/1-UlfHukW.il
1 kl?lfu/*/< )
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Plate (til)
© Count
0A
G
® Count
G
® Count
G
© Count
§b
G
® Count
Si B
G
43 44
45
n
46 50
51 54
55 58
59
~
60 63
64
~
65 6a
69
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70 73
74
~
75 78
79
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0
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11 MM
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1 1 7l
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1 1 1
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~
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Mill
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~
llll
~
II1II
~
II II
~
II II
~
0
1 11
~
III Ull
LLIJJ
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~
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~
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~
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0
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1
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~
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~
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~
Ml 1
~
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~
1 1 1 II 1 1
Mill
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~
Mil
~
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n
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~
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~
0
111
~
111 11II
Mill
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~
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~
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~
0
in
~
II1 Ull
Mill
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~
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~
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~
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~
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Mill
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~
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111
~
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Mill
~
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III 1
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0
-------
WORKSHEET FOR THE SPRM -^AA RECOVER* STUDY IN THE AMES ASSAY
IN STRAIN TA98
Extraction Spiked Dose
Method ( ug/1.5 L water )
* Recovery
2\ S-9 Mix 10* S-9 Mix
t~> (yCfi^Z. fcT(XL
j?H /'
3-SV
pH .2. -/rrntu-vv
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2400
RECOVERY EFFICIENCY
2-Aminoanthracene Dose-Response
leoo _
1200 _
600 _
2X S9
SYMBOLS
UNETYPE
y~a+b*x
n=18
o=146.710B
b=4233. 4329
sm-121.2130
s=34.1785
9^=163. 1250
r=0.9883
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216
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237
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322
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1000
684
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1 000
723
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2157
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0000
5000
_ 1 0
0000
ug/plate 10/25/85
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2400
RECOVERY EFFICIENCY
250 ug 2AA/1.5 1 Blank, Base/Acid
180D _
I ZOO _
/
600 _
21 S9. pHll fraction
SYMBOL0 «
LINETYPE
y=a+b*x
n=»18
0=171.9618
b=3664. OBI 4
s =133.2100
r1
s =37. 4502
sk=178.7993
r=0.9815
ug/platG
10/25/85
1
I •
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0000
4 5
00
IJ
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rz
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00
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47
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-------
HERL IN VITRO RESUL TS FORM
~
}4
5 a
17 V R
System ID
Research
Lab ID
9 14
A4?bbU
MO DA YR
Experiment Dale
®6«
15 18
19 20
21 24
LAB YR NUMBER
Test Sample Identification
25 30
H
Activation Batch
t
31 32
Test Type
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33 39
39 42
T02Z00H3Z
} Strain Batch No
0 711Q 72 73 74 75
fe m m
Microorganism
QD 76 78
Animal
A
Organ
L
Inducer
A
¦ 2% S? Mix
Htmarkt
©«5
QPhenocopy
Check
Conclusion
\table 131
©66
P"I Sterility
11 | S 9 Mil
I Not Contam
67 70
he
Mctivation
71
Sample
Sterility
Check
I Not Contam tune (mini Temp (Cent/, technician
DoseLevei
Plate A
Plate B
Plate C
Plate D
Plate E
Card
Code
®
Solvent
Positive
(table 11)
Q Units of
Concentration
Blank - mg/m/
2 ug/rnf
0
Stock con-
centration
®
Ami Per
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Count
©
B
G
®
Count
®
B
G
©
Count
®
B
G
®
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B
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0
Count
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B
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43 44
SOL
45
~
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5/ 54
11 kkl
55 58
11 kkl
by
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0
11 kkl
1
70 73
Mill
74
u
Mill
A
p"s kkl
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11 kkl
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Mill
~
0
0
0
0
0
0
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0
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~
11 MloW
11 kkl
I H/ki
~
1 kkkl
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1 kkkl
1 1
11/kM
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1 1 NU.,1
11 kkl
1 If klfl
~
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1 1
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11
Mill
~
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11 kll/M
11 kkl
1kkkl
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u
Mill
~
kkl
~
1 1 MUM
11 kkl
kkklrl
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-------
HERL IN VITRO RESUL TS C0N7INUA 7ION FORM
ro
-o
ft
Q ^
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5 8
9 14
\c\pii\n
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System
ID
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Lab ID
MO DA YR
Experiment Date
15 18 19 20 21 24
Mi/j/yui-rfm-iaiVioiJi
Lab YR Number
Test Sample Identification
i hjV/um
lActivation Batch
1 1% j> 7 At?K
31 32
02
Test Type
(Table 10)
33 38 39 42
I l \TlA\ns\ U|j |<->171
f Strain Batch No
Microorganism
2) Solvent
Positive
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(Z) Units ol
Loncentralion
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2 - w ml
Dose Level
Stock Con
cent/atton ,
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Plate (yl)
Plate A
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&>B
G
Plate B
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G
Plate C
® Count
©5
G
Plate D
® Count
®B
G
Plate £
®Count
G
Card
Code
43 44
45
n
}fso
51 54
55 58
59
~
60 63
64
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65 68
69
~
10 73
74
~
75 78
79
~
80
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CD
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1400
RECOVERY EFFICIENCY
2-Aminoanthracene Dose-ResponsG
1050 _
700 _
i
350 _
10X S9
SYMBOL0©
LINETYPE
y=a+b*x
n=17
aa19.0050
b=2342.3229
33.5711
s
M
g,=9.7B23
9k=45. 4060
r=0.9972
0.6
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0
0O0U
4 1
0000
0
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4 7.
0000
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49
0000
0100
77 .
0000
0100
61 .
0000
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91
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02-50
97
0000
0250
73
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. O500
107
0000
0500
1 15.
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0500
105
0000
1000
201 .
0000
1000
213 .
0000
1000
195.
0000
500'.'
1 1 .-9
0000
500'.'
1 2u5.
0000
5000
1 !.<>.
0000
ug/p1 ate
10/25/85
-------
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CD
1000
RECOVERY EFFICIENCY
250 ug 2AA/1. 5 1 Blank. Base/Acid
750 L
500 L
250 L
0.0
0. 6
ug/p1 ate
10/25/85
10* S9.
SYMBDL=
LINETYPE= —
y=a+b*x
n=18
a=39.3045
b=l493.6831
s, =15. 8439
pHl1 fraction
s0=4. 4543
sb=21.2663
r=0.9984
I •
\ 1
¦ I ¦
0
0 0 0 0
4?
it fi o n
o
000 U
4 £
(.lOru'i
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0 0 0 0
nt'1
0 00 0
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4 3
MfUjO
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0 0 i~t 0
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t-7
fi 0 i"> 0
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0 0 l"i 0
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n
0 0 0 0
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0 0 fi 0
0*500
1 07
fi nfiO
0 j 0 fl
101
l J M IJ 0
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1 000
r,oon
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GO
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1000
RECOVERY EFFICIENCY
250 ug 2AA/1. 5 1 Municipal. Acid/Base
750 _
500 _
250 _
102 S9. unsplhed municipal
SYMBOL®o
LINETYPE-
y°a+b«x
re21
a-47.8672 8=1.9720
b=4.0431 s„=1.1315
a "1.0396 r=0.6340
10X S9, spiked municipal
SYMBOL0 o r
LINETYPE
y°a+b»x
n-21
a=28.7590
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I
~
II 1 1
~
Mil
~
0
m
~
II 1 III
II 111
1
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II 1
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n
1 1
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~
0
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~
1 1 1 U 1
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1
n
1 1
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I
~
II 1
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MM
~
0
m
~
II 1 III
1 1 111
1
n
1
~
I 11
n
II 1
n
IMII
n
0
-------
WORKSHEET FOR THE SPRM RECOVERY STUDY IN THE AMES ASSAY
IN STRAIN TA100
Extraction
Method
Spliced Dose
( ug/1.5 L water )
-S9
* Recovery
fauz/Actt $v0° /
^ Ua+J'Is ia?x£as °y*60yo<$v}j
*&>0 14-U-
v. tke
^000 6
£n "f"/\6 ^ /~) D &
E-296
-------
M
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.—I
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w
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+->
L
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DC
O
CD
480
RECOVERY EFFICIENCY
4-Nitrobenzoic Acid Dose-Response
360 _
240 _
120 1
560
-S9
SYMBOL—
LINETYPE= —
y=a+b*x
n=18
a=142. 1309
b=0.5039
sr =25.1906
s.=7.8270
st=0.0336
r=0. 9662
o
o
0
10
10
10
50
50
SO
1*6
100
100
250
250
£50
500
500
500
I >
0090
0000
0000
0000
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0000
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0000
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148
15?
181
142.
102
118
163
181 .
151
213
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181
253 .
276
325
371
407 .
382
I >
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
Wg/plate
12/17/85
-------
400
RECOVERY EFFICIENCY
5000 ug 4NBA/1.5 1 Blank, Acid/Base
300 _
200 _
100
-S9
SYMBOL^
LINETYPE= —
y=o+b*x
n=»18
a=154.1017
b=0. 4012
sM=24. 3090
s.=7.5530
sfc=0.0324
r=0.9514
140
260
420
560
y.f
o
e
U
10
10
10
50 .
50
50
100
100
100
250
250
250
500.
500
500
I >
0000
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Y <
169
158
37
141
153
161 .
153
130
183
180
210.
231 .
269
266
275.
383
326.
324
I >
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
OPI00
0000
ug/plate
12/17/85
-------
m
i
NJ
KD
ai
4->
o
t i
Q_
\
(/)
4->
C
0
+J
(_
Q)
>
01
(H
a
o
360
RECOVERY EFFICIENCY
5000 ug 4NBA/1. 5 1 Blank. Acid/Base
270
180 _
140
560
-S9
SYMBOL-a
LINETYPE—
y°a+b*x
na19
a=155.3002
b=0.3008
sM=20.7226
ug/p1atQ
12/17/85
o
o
O
10
10
10
10
50
50
50
100
100
100
250
250
250
500
500
500
s,°6.1638
sh=0.0272
r=0.9370
: i
0000
0000
0000
0000
0000
0000
0000
.0000
0000
0000
0000
0000
0000
0000
000W
0000
0000
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0000
Y
169
158
97
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173
157
160
155
173
193
188
193
194
235
249
228
337
275
290
< I >
0000
0000
0M00
0000
. 0000
0000
. 0000
0000
0000
.0000
0000
0000
0000
0000
0000
0000
0000
0000
0000
-------
HERL IN VITRO RESUL TS FORM
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} i
5 8
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cn-it-
System ID
Research
Lab ID
9 >4
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MO DA YR
Experiment Date
15 18
19 20
21 24
LAB YR NUMBER
Test Sample Identification
n
25 30
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t
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67 70
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43 JT
501 03
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0 Unnso/
Concentration
Slant - mg/ml
2 " tig/m!
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centration
w/rf'h)
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it &4 "
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()
forms Completion]
Initials [ | | | I
-------
HERL IN VITRO RESUL TS CONTINUA HON FORM
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31 32
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33 3a 39 42
i \tum\o\o\ imi/i/i
em\ Research
Lab ID
MO DA YR
Experiment Date
Lab YR Number
ist Sample Identification
^Activation Batct
Test Type
(Table 10)
f Strain Batch No
Microorganism
Dose Level
Plate A
Plate B
Plate C
Plate D
Plate E
Card
Code
0 Solvent
Positive
(Table 9 tf
(j) Units ot
Concentration
Blank - mg/ml
2 - pg/ml
© Stock Con
centrahpn,
(M4 /pure )
© Amt Per
Plate (t/ll
S Count
®A
G
® Count
Qb
G
® Count
(§>£
G
S) Count
^B
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® Count
G
43 44
45
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46 50
51 54
55 5a
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65 68
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70 73
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75 78
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blilollol 1
1 1 \to
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AM —
1 l/HM 1
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1 1/
-------
HERL IN VITRO RESUL TS CONTINUATION FORM
t
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9 14
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1/1^1/171.11^1
System
Research
MO DA YR
& *
*
ID
Lab ID
Experiment Date
15 18 19 20 21 24
Lab YR Number
Test Sample Identification
25 30
i ill i i
f Activation Batch
3I 32
03
Test Type
17 able 101
33 38 39 4 J
i i7ui/iJioi ui'fi/ m
f Strain Batch No
Microorganism
Dose Level
Plate A
Plate B
Plate C
Plate D
Plate E
Card
Code
(ft) Solvent
Positive
(Table 11)
0 Units of
Concentration
Stank - mg/mt
2 - itg/mt
© Stock Con
cent ration
/t,Ult J
© Amt Per
Plate (pi)
^ Count
®B
G
® Count
G
® Count
G
® Count
®B
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©A
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43 44
m
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St 54
55 58
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60 63
64
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65 68
69
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70 73
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1 bbIM |
II ^3
11/42
Cz.^2
Cz.^2
Mill
Mill
m
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~
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1 1 MM
1 m
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~
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1 1 W
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~
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m
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liiolollol
1 1 Uol
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0
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1 1 llv
~
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~
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0
m
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111 111
1 1 111
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0
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1 1 111
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0
1 1
~
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~
III 1
~
Mil
~
0
-------
APPENDIX F
STRAIN FUNCTION, CELL TITER, AND CELL VIABILITY RECORD
For the purpose of quality control of the Ames assay, the bacterial strains
used were checked for their genotype functions. These characteristics were analyzed
for each experiment. The cell titer and cell viability were measured whenever an
experiment was performed. The record of these analyses and measurements is
attached to this report as Appendix F.
F-303
-------
STRAIN FUNCTION TESTS, CELL TITER AND VIABILITY RECORD
Experiment Date ^ / J / 55
Initial J~i-l C
Test
TA98
TA100
His"'' Bio
His+ Bio"
+ 1
-f
¦f
i
-h
His Bio
His" Bio
Ampicillin Sensitivity^"
Crystal Violet Sensitivity*
(Inhibition Zone, mm)
2
UV Sensitivity, 9 sec.
(Irradiated/non-irradiated)
Negative Control"*:
Spontaneous Mutation
Positive Controls :
1)
2)
3)
2I
30
3 3
2-Nitrofluorene (TA98, -S9, ug/plate)
1) H'4
2) /U*
3)
2-Ammofluorene (TA98, +S9, / jug/plate)
1) CfO 33}
2)^i"
D$n
(S/ (/
-/Hi
1)
2)
3)
/5°
JS2
O.D. Reading for 10-1 Dilution
Viable Cell Count ( x 10^ cells/ml) /, f
Sodium Azide (TA100, -S9,'j..fjug/plate)
1) pf
2)
3)
2-Aminoanthracene (TA100, +S9,->.Jjug/plate)
1) 2&1 /<>¦>¦:
2) m
3) J7¦>uf
(j%±7) (/"V-y,))
O,
J. 2
4 = Growth, no inhibition zone.
- = No growth, record the diameter of the inhibition zone, in mm.
+++ = Full growth, non-irradiated control.
++ = 10"^ to 10"* x control.
+ = 10 to >0 x control.
- = No growth.
Plate count, revertants/plate.
F-304
-------
STRAIN FUNCTION TESTS, CELL TITER AND VIABILITY RECORD
Experiment Date ? /2 2 / $5'
Initial KLC
Test
TA98
TAIOO
His+ Bio+ 1
His+ Bio"
His" Bio+
His" Bio
Ampicillm Sensitivity^
Crystal Violet Sensitivity*
(Inhibition Zone, mm)
2
UV Sensitivity, 9 sec.
(Irradiated/non-irradiated)
Negative Control^:
Spontaneous Mutation
i
/i
-/JH
1) 24
2) J ?
3) jq
Positive Controls :
2-Nitrofluorene (TA98, -S9, ug/plate)
1)
2) /Sl$
3) /5SS
2-Aminofluorene (TA98, +S9, / ug/plate)
1) J08
2) ^S2 Jio
3) *12
t
i
;7
i/JH
1) /7°
2)
3> /H
O.D. Reading for 10~ Dilution
Viable Cell Count ( x 10^ cells/ml)
0.^1
2^ 0
Sodium Azide (TA100, -S9,->^jug/plate)
1) HI
2) 3 7o
3J ni
2-Aminoanthracene (TA100, +S9,-1 5"jug/plate)
1) JM] 116
2) J30
3) f$/
(j 'A Si) (/a /. •/))
O, 32
J, /
^ 4 = Growth, no inhibition zone.
- = No growth, record the diameter of the inhibition zone, in mm.
2
+++ = Full growth, non-irradiated control.
++ = 10"^ to 10"^ x control.
+ = 10 to >0 x control.
- = No growth.
^ Plate count, revertants/plate.
F-305
-------
STRAIN FUNCTION TESTS, CELL TITER AND VIABILITY RECORD
Experiment
Date 2 / 65
Initial
KIC
Test
TA98
TAlOO
His+ Bio+ 1
His+ Bio"
His" Bio+
His- Bio~
Ampicillm Sensitivity^"
Crystal Violet Sensitivity*
(Inhibition Zone, nun)
2
UV Sensitivity, 9 sec.
(Irradiated/non-irradiated)
Negative Control^:
Spontaneous Mutation
i
t
n
-/+H
1)
2)
3)
¦J>o
Positive Controls :
2-Nitrof luorene (TA98, -S9, ug/plate)
1) nil
2) /1JO
3) )045
2-Aminofluorene (TA98, +S9, / ug/plate)
1)311
2) 283
j 7o
O.D. Reading for 10~* Dilution
Viable Cell Count ( x 10^ cells/ml) /, £
t
¦f
1)
2)
3)
n
-/Hi
m
m
/ u
Sodium Azide (TAlOO, -S9,j jjug/plate)
1) 7 > 1
2) U3
3) £51
2-Aminoanthracene (TAlOO, +S9 ^jug/plate)
1)^6 76 f$/o
2) 7J jjf$2
3) J7/2 W5S
o^2
2.5
+ = Growth, no inhibition zone.
- = No growth, record the diameter of the inhibition zone, in mm.
+++ = Full growth, non-irradiated control.
++ = lO'l to 10~2 x control.
+ = 10 to >0 x control.
- = No growth.
Plate count, revertants/plate.
F-306
-------
STRAIN FUNCTION TESTS, CELL TITER AND VIABILITY RECORD
Experiment Date S//J/ St
Initial
KZC
Test
TA98
TA100
HlS+ BlO+ 1
His Bio"
i
i
His Bio
His Bio
Ampicillin Sensitivity
Crystal Violet Sensitivity^
(Inhibition Zone, mm)
2
UV Sensitivity, 9 sec.
(Irradiated/non-irradiated)
Negative Control^:
Spontaneous Mutation
1)
2)
3)
2 1
~/Hi
21
Si
2 I
Positive Controls :
2-Nitrofluorene (TA98, -S9, "Vug/plate)
1)
2) /$1\
3) H fo
2-Aminofluorene (TA98, +S9, / ug/plate)
1) 2*38
2)
3) -**75
O.D. Reading for 10~^ Dilution Qt -V 7
Viable Cell Count ( x 10^ cells/ml) jt ^
1)
2)
3)
Sodium Azide (TA100, -S9, ,ug/plate)
1)
2)
3)
2-Aminoanthracene (TA100, +S9, /jg/plate)
1)
2)
3)
+ = Growth, no inhibition zone.
- = No growth, record the diameter of the inhibition zone, in mm.
+++ = Full growth, non-irradiated control.
++ = 10-1 to x controi_
+ = 10 to >0 x control.
- = No growth.
Plate count, revertants/plate.
F-307
-------
STRAIN FUNCTION TESTS, CELL TITER AND VIABILITY RECORD
Experiment Date J / J / J f
Initial
K1C
Test
TA98
TA100
HlS+ BlO+ 1
His+ Bio"
His" Bio+
His" Bio
Ampicillin Sensitivity1
Crystal Violet Sensitivity1
(Inhibition Zone, mm)
2
UV Sensitivity, 9 sec.
(Irradiated/non-irradiated)
Negative Control3:
Spontaneous Mutation
Positive Controls"
-t
1
n
-/m
1)
2) JO
3) 32
i
2-Nitrofluorene (TA98, -S9, ilg/plate)
1) /oSS
2) ///3
3) /ouf
2-Aminofluorene (TA98, +S9, / ug/plate)
1)
2) 473 W(»
3)
(j% s?)
O.D. Reading for 10~* Dilution
Q
Viable Cell Count ( x 10 cells/ml) 2. O
u
-/Hi
1) 3^7
2)
3) 3/J
Sodium Azide (TA100, -S9,^Aig/plate)
1) f3iS
2) /*?!
3) /3*(
2-Aminoanthracene (TA100, +S9,J'j jug/plate)
1) JjJS I nh
2) /Jj3
3) ->7/; /,-})
CM*/)
O.st
3.2
1 + = Growth, no inhibition zone.
- = No growth, record the diameter of the inhibition zone, in mm.
2
+++ = Full growth, non-irradiated control.
++ = 10~, to 10~2 x control.
+ = 10 to >0 x control.
- = No growth.
3 Plate count, revertants/plate.
F-308
-------
STRAIN FUNCTION TESTS, CELL TITER AND VIABILITY RECORD
Experiment Date */ /
Initial
xir
Test
TA98
TA100
His"1" Bio
His+ Bio"
His- Bio+
+ 1
1
i
1
1
His Bio
Ampicillin Sensitivity1
Crystal Violet Sensitivity1
(Inhibition Zone, mm)
UV Sensitivity, 9 sec.^
(Irradiated/non-irradiated)
Negative Control3:
Spontaneous Mutation
Positive Controls :
n n
-/m -/m
1) 35 l) /3 /
2) if 2) /id
3) ^ 3) / )0
2-Nitrofluorene (TA98, -S9, ug/plate)
1) //ss
2) A* 7
3) /.j/O
2-Aminofluorene (TA98, +S9, / ug/plate)
1) zii 462
2) di0
M 351
O.D. Reading for 10-1 Dilution o, -V /
Viable Cell Count ( x 109 cells/ml)
Sodium Azide (TA100, -S9,JjTig/plate)
/$yi
2) U41
3) /7^y
2-Aminoanthracene (TA100, +S9,-'5xig/plate)
1) jf3S /^J>7
2) J Hi
3) J-2 /jj-
<}< 3 $
* 1
1 + = Growth, no inhibition zone.
- = No growth, record the diameter of the inhibition zone, in mm.
2
+++ = Full growth, non-irradiated control.
++ = 10"1 to 10-2 x control.
+ = 10 to >0 x control.
- = No growth.
^ Plate count, revertants/plate.
F-309
-------
STRAIN FUNCTION TESTS, CELL TITER AND VIABILITY RECORD
Experiment Date / 0 / 4 / S$
Initial KZ C
Test
TA98
TA100
His+ Bio+ 1
His Bio"
His Bio
His Bio
"f
Ampicillin Sensitivity
Crystal Violet Sensitivity*
(Inhibition Zone, mm)
UV Sensitivity, 9 sec.^
(Irradiated/non-irradiated)
Negative Control^:
Spontaneous Mutation
20
1) n
2) 34
3)
Positive Controls :
2-Nitrofluorene (TA98, -S9, 4 -ug/plate)
1)
2) Hi
3) g$2
2-Aminofluorene (TA98, +S9, / Aig/plate)
1) 315
2)
3) 342 ^7
CJ%51) (/*%$)
i
+
/ 7
-t/Hi
1) /s /
2) M2>
3) J/j
O.D. Reading for 10~ Dilution
Viable Cell Count ( x 10 cells/ml)
0,4$
/.$
Sodium Azide (TA100, -S9,-*$;ug/plate)
1)
2) 3SI
3) 351
2-Aminoanthracene (TA100, +S9,<>.£ug/plate)
1) /o)i
2)
3) /S*5 /JS7
(1M) (/«¦;,,))
o, 3S
IA
4 = Growth, no inhibition zone.
- = No growth, record the diameter of the inhibition zone, in mm.
^ +++ = Full growth, non-irradiated control.
++ = 10"! to 10~2 x control.
+ = 10 to >0 x control.
- = No growth.
^ Plate count, revertants/plate.
F-310
-------
STRAIN FUNCTION TESTS, CELL TITER AND VIABILITY RECORD
Experiment Date
a / n! is
Initial /KI C
Test
TA98
TA100
His+ Bio+ 1
His+ Bio"
His- Bio+
His- Bio
Ampicillin Sensitivity^
Crystal Violet Sensitivity^
(Inhibition Zone, mm)
2
UV Sensitivity, 9 sec.
(Irradiated/non-irradiated)
Negative Control3:
Spontaneous Mutation
Positive Controls"
¦+
/7
1) 3H-
2) JO
3) 3d
2-Nitrofluorene (TA98, -S9, ug/plate)
1) /lit
2)
3> //S°
1
-f
H
-/Hi
l)
2> U1
3) /si
Sodium Azide (TA100, -S9,«,i;ug/plate]
1) Mil
2) /Mi
3) H*>}
2-Aminofluorene (TA98
, +S9, I Aig/plate) 2-Aminoanthracene (TA100, +S9,-*5"Jug/plate)
i) ^ d /K7 f-fi
2)
3>V71 M7
(S/,si) (/>**?)
O.D. Reading for 10~* Dilution
Viable Cell Count ( x 109 cells/ml) /, 2
2) J Ml Ui
3> /?35 m
o,$£
/•I
* •* = Growth, no inhibition zone.
- = No growth, record the diameter of the inhibition zone, in mm.
2
+++ = Full growth, non-irradiated control.
++ = to 10 x control.
+ = 10 to >0 x control.
- = No growth.
^ Plate count, revertants/plate.
F-311
-------
STRAIN FUNCTION TESTS, CELL TITER AND VIABILITY RECORD
Experiment Date /° / 1 f / J $
Initial j-{± C
Test
TA98
TAIOO
His+ Bio+ 1
His+ Bio"
His- Bio+
His" Bio~
Ampicillin Sensitivity*
Crystal Violet Sensitivity*
(Inhibition Zone, mm)
2
UV Sensitivity, 9 sec.
(Irradiated/non-irradiated)
Negative Control3:
Spontaneous Mutation
1
/ S
-/Hi
1) 1$
2) d4
3) JS
Positive Controls :
2-Nitrofluorene (TA98, -S9, ^ag/plate)
1) /o3$
2) /oj>2
3) /ill
2-Aminofluorene (TA98, +S9, ) ug/plate) .
1)471 J?1
2) 47$
3) 464
4
i
n
-/Hi
l) /41
2> /Si
3> /JJ
O.D. Reading for 10" Dilution 0,4-5
Viable Cell Count ( x 10^ cells/ml) /0
Sodium Azide (TA100, -S9,->.fug/plate)
1) 311
2) S/O
3) sC4
2-Aminoanthracene (TA100, +S9 ,J,_£ug/plate)
1) l"J°1
2) j Jo j /jjj
3) JS Jl fi o7
( ) ( /u ^
0,3 7
1.7
* + = Growth, no inhibition zone.
- = No growth, record the diameter of the inhibition zone, in mm.
2 +++ = Full growth, non-irradiated control.
++ = 10"1 to 10~2 x control.
+= 10 2 to >0 x control.
- = No growth.
^ Plate count, revertants/plate.
F-312
-------
STRAIN FUNCTION TESTS, CELL TITER AND VIABILITY RECORD
Experiment Date
Jf /ix /it
Initial /¦< Z C
Test
TA98
TA100
His+ Bio+ 1
His+ Bio"
His" Bio+
His" Bio"
Ampicillin Sensitivity*
Crystal Violet Sensitivity1
(Inhibition Zone, mm)
UV Sensitivity, 9 sec.^
(Irradiated/non-irradiated)
Negative Control3:
Spontaneous Mutation
Positive Controls"
-i
¦+
2 I
-/H-f
1) 3 7
2) 33
3) 31
2-Nitrofluorene (TA98, -S9, ^-fug/plate)
1) //$&
2) /til
3) /lU
2-Aminofluorene (TA98, +S9, J ug/plate)
1) 81$ ill
2) &n jo/
3) 8is 111
+
+
ll
~/i4i
Wii) (/«%*?)
O.D. Reading for 10" Dilution
Viable Cell Count ( x 10 cells/ml)
oAC
hi
1) /Jj
2) /H
3) /S6
Sodium Azide (TA100, -S9^^g/plate)
1) m
2) no
3) (Si
2-Aminoanthracene (TA100, +S9^J ^ug/plate)
1) sfo
2) 22S') 113
3) 1171 JJ?
o,56
¦?. I
+ = Growth, no inhibition zone.
- = No growth, record the diameter of the inhibition zone, in mm.
2
+++ = Full growth, non-irradiated control.
++ = to 10~2 x control.
+ = 10 to >0 x control.
- = No growth.
3 Plate count, revertants/plate.
F-313
-------
STRAIN FUNCTION TESTS, CELL TITER AND VIABILITY RECORD
Experiment Date /-? / / 7 !&S
Initial
AIL
Test
TA98
TA100
His+ Bio+ *
His Bio"
+
-f
-f
His Bio
His" Bio
Ampicillin Sensitivity*
Crystal Violet Sensitivity*
(Inhibition Zone, mm)
2
UV Sensitivity, 9 sec.
(Irradiated/non-irradiated)
Negative Control^:
Spontaneous Mutation
Positive Controls :
//
-/Hi
1) 3H-
2) Jd
3) J 7
2-Nitrofluorene (TA98, -S9, jug/plate)
1) /°1 >
2)/°SS
3)//oS
2-Aminofluorene (TA98, +S9, / ug/plate)
1) 715
2) 731 iSl
//
i) m
2> /;/
3) /S7
Sodium Azide (TA100,
1) 686
2) 656
3) 0°
-S9,<>. j";ug/plate)
O.D. Reading for 10"1 Dilution
Viable Cell Count ( x 10^ cells/ml)
J,-?/
/¦S
2-Aminoanthracene (TA100, +S9,->.f jug/plate)
1) /Ud 1U
2) >73
3) loS3 I
0,3d
J. J
+ = Growth, no inhibition zone.
- = No growth, record the diameter of the inhibition zone, in mm.
+++ = Full growth, non-irradiated control.
++ = to 10~2 x control.
+ = 10 to >0 x control.
- = No growth.
Plate count, revertants/plate.
F-314
-------
STRAIN FUNCTION TESTS, CELL TITER AND VIABILITY RECORD
Experiment
Date 2 / // / H
Initial R. 1, C,
Test
TA98
TA100
His+ BlO+ 1
His+ Bio"
His Bio
His" Bio"
-i
-f
Ampicillin Sensitivity
Crystal Violet Sensitivity1
(Inhibition Zone, mm)
2
UV Sensitivity, 9 sec.
(Irradiated/non-irradiated)
Negative Control3:
Spontaneous Mutation
Positive Controls :
2-Nitrofluorene (TA98, -S9,
n
-/ + H
1) 3S
2)
3> 28
ug/plate)
1)
2)
3)
1)
2)
3)
Sodium Azide (TA100, -S9, jug/plate)
1)
2)
3)
2-Aminofluorene (TA98, +S9, / ug/plate)
1) )42
2) /oC
3> Jo-j
n
2-Aminoanthracene (TAMO, +S9piP,ug/plate)
1) 265
2) 23S
O.D. Reading for 10"1 Dilution
S-1 HU)
(3 j /, 5-7/1?*)
Viable Cell Count ( x 10 cells/ml)
0-45
/.2
+ = Growth, no inhibition zone.
- = No growth, record the diameter of the inhibition zone, in mm.
2
+++ = Full growth, non-irradiated control.
++ = to 10" x control.
+ = 10 to >0 x control.
- = No growth.
3 Plate count, revertants/plate.
F-315
-------
STRAIN FUNCTION TESTS, CELL TITER AND VIABILITY RECORD
Experiment Date 2
/ is he
Initial
Test
TA98
TA100
His+ Bio+ 1
His+ Bio"
His- Bio+
His" Bio~
i
"f
Ampicillin Sensitivity
Crystal Violet Sensitivity*
(Inhibition Zone, mm)
2
UV Sensitivity, 9 sec.
(Irradiated/non-irradiated)
Negative Control3:
Spontaneous Mutation
Positive Controls :
2-Nitrofluorene (TA98, -S9,
20
-/Hi
i) 3 I
2> 3 I
3) 2$
ug/plate)
1)
2)
3)
1)
2)
3)
Sodium Azide (TA100, -S9, jug/plate)
1)
2)
3)
2-Ammofluorene (TA98, +S9, j ug/plate)
1) 3$$ D 81
2) JJ/ 2) S2
J) 37$
O.D. Reading for 10 Dilution d-$jCf
18
+S9,3£ug/plate)
3) M
(3°/>
2-Aminoanthracene (TA]
i
2)J/$
3 ^ / "? 1 Cckns? <¦/<'¦¦ih'iUI of (_ e /¦>.
(39 7* 5-f W a)
on ->/*- <
of fa p!
Viable Cell Count ( x 10 cells/ml)
/- I
+ = Growth, no inhibition zone.
- = No growth, record the diameter of the inhibition zone, in mm.
2
+++ = Full growth, non-irradiated control.
++ = io"J to 10~2 x control.
+ = 10~ to >0 x control.
- a No growth.
^ Plate count, revertants/plate.
F-316
-------
APPENDIX G
PRIMARY DATA WORK SHEET FOR STATISTICAL ANALYSIS
For the protocol validation study, several statistical analyses were performed
to establish the method background. The study included both the sample preparation
and the Ames assay procedures. Therefore, three negative controls were involved:
(1) the revertant counts of the blank water extract representing the laboratory, field,
travel, sample preparation method, and mutagenicity testing procedure blanks; (2)
the revertant counts of the DMSO solvent vehicle control in the Ames assay; and,
(3) the spontaneous mutation revertant counts as the blank control for the mutagenicity
measurement of the bacterial strain. An F-test with two factor block analysis of
variance was used to analyze the similarities among these three controls. If there
was any difference among these controls, a paired t-test was used to find out where
the difference was. The primary data and calculation processes are recorded on the
work sheets which are attached to this report as Appendix G.
G-317
-------
WORK SHEET
FOR STATISTICAL ANALYSIS
TA98, —
S-9
a= 8 , b=3,
n=3
y^d
Exp. Date
DMSO
SR
WATER
*1
7/22/85
2&
27
35
21
8/2/85
27
28
33
21
8/13/85
—
—
—
—
8/23/85
21
31
38
9/24/85
2$
21
3i
21
10/4/85
23
J>o
3i
30
10/17/85
26
3+
32
10/25/85
32
3/
J I
11/22/85
28
3$
34
12/ 17/85
—
—
21
30
jS
Y= 3 I
(i
Days (D) SSa= L ( Y. ' T ) 3*3 (4 * * * « < <* "> ' 4 ? ) - ? * H z 251
Treatments (T) SSb= tin L ( y7 - T^*" 3 ^ ' 4 /^ ) z 112
D x T SSah= „ r > ( Y;.J - f; - Yl f ' ') 2 3(^1 J ¦ Ji
Residual
F-test:
J-1
SSab= n I L (y;
l-l J-i
_ & i. i
ssv=
t -ILL ( Yi]k - Yn) /oW
;-/ j-/ /-/
SS
df
(a-1)
MS (SS/df)
F(MS/s2)
Tabulate («*
D
2di
7
J3
/. i"
1 22
(b-l)
T
712
j>H
Ji.o
3. 2
(a-1)(b-l)
D x T
3L3
2(,
/.2
/.u
ab(n-1)
Residual
/oH
*8
22 (s2)
t-test;
DMSO vs. SR
DMSO vs. WATER
SR vs. WATER
t Tabulate (•<= 0.05)
H /, m
-^7? /.j1$
/J]$
Ha
6/^ 7 9/-15 0
J-jiO 7 PslSo
j-ij o 7 Sf\
G-318
-------
WCRK SHEET FOR STATISTICAL ANALYSIS
TA98, 2 /jS-9
a= £j_, b=3, n=3 Y
Exp. Date DMSO
'i]
SR
WATER
7/22/85
8/2/85
8/13/85
8/23/85
9/24/85
10/4/85
10/17/85
10/25/85
11/22/85
12/ 17/85
33
33
5 I
40
jl
J7
45
41
*1
4S
34
3?
J1
S4
45
41
44
45
J5
45
41
4i
4S
si
50
51
Si
40
44
31
3l
41
42
*6
4S
41
So
Y= Uflf
Days (D) SSa= J,n Lf ( Yf " 9^- (W) ; Ml
Treatments (T) SSb= an £ ( 9] ~ Y^' ^ '• ll°l
J' I
U h
d x t ssab= nil ( Yij ' >7 " Yj 71 Y ) : ^(4? 7) - /*?? I
Residual
F-test:
ssr= iii cy;jk- Yr,f: 'W
; i j-i f-1
SS
df
(a-1)
MS(SS/df)
F(MS/s2)
Tabulate (•*
D
/i£1
s
3./S
J./Jf
(b-1)
T
11*7
2
SS4
//. °S
J./7
(a-1)(b-1)
D x T
/*?/
fl
n
/Ji
/H
ab(n-1)
Residual
j Jo4
$4
So (s2)
t-test;
t
Tabulate (*=
0.05)
ha
DMSO vs. SR
-1.14
/.m
DMSO vs. WATER
-J. S5
/, S15
J4.0 7 9s>"JS0
SR vs. WATER
-/. 11
/.S1$
HiO 7 Sf\
G-319
-------
WORK SHEET FOR STATISTICAL ANALYSIS
TA98, /->Zs-9
a=_2_, b=3, n=3 Y
Exp. Date DMSO
SR
WATER
7/22/85
8/2/85
8/13/85
8/23/85
9/24/85
10/4/85
10/17/85
10/25/85
11/22/85
12/ 17/85
40
41
f-3
47
4$
4L
4$
4i
46
47
43
41
41
*1
48
4,$
41
41
Si
4L
S4
S3
i-<5
SI
43
43
44
50
41
41
51
4 7
46
*j 44 46 $ I y= 41
Days (D) SSar&n^ ( ' f f " 1 ( ) ' 7°-<
Treatments (T) SSb= (Yj~Y)'': j7(j^) ' 75 3
D x T
Residual
F-test;
SSab=
SSr=
j'l
ah
tl 4 ^
111 ( Ynk
r-i j i x-t
<7 " Tt f 7 ^/J
- y, j )': if**
SS
df
MS(SS/df)
F(MS/s2)
Tabulate
(a-1)
D
7*z
5
n
U1
i.u
(b-1)
T
783
Z
3U
7S4
3/7
(a-1)(b-1)
D x T
S/3
/(
32
O.tZ
/J4
ab(n-1)
Residual
S4
Si (S2)
t-test:
DMSO vs. SR
DMSO vs. WATER
SR vs. WATER
t
-o.tf
-s.a
-J 51
Tabulate («<= 0.05)
I. his
I, SIS
/ t;s
ha
PMSv *¦ S/i
/-Lo > \)Mso
Hjo 7 5/^
G-320
-------
APPENDIX H
PRIMARY DATA WORK SHEET
FOR QUALITY CONTROL CHART CALIBRATION
Quality control charts were constructed for the method background to provide
graphic assessment of accuracy and precision for each analysis and to provide instant
detection of unacceptable data. The calculation process for establishing these data
are recorded on the work sheets which are attached to this report as Appendix H.
The calculated mean value (X) was assumed as the true value for accuracy evaluation.
The range (R) chart was used for precision measurements. The upper control limit
(UCL) and lower control limit (LCL) represented the 99% confidence interval.
H-321
-------
WORK SHEET FOR DATA QUALITY CONTROL CHART
Strain TA98 Sample /~fj O Activation ~~-5 "J
Exp. Date Plate Count Average SD
(1) (2) (3) X C
Variance
(T4
cv
r
— X 100%
K
Range
Total n= f
Zx= J/4
ZR= 71
1. X = H f n =
2. ucL- = x + cl- = jy, ? f % o - -y3, ;
3. LCL- = X - CL- = 34.J ~ 1,0 : <]
4. R = 2R t n = J, &
5. UCL- = D4 x R = J,$7SXt,$ ;
6. LCL= = D, x R = 0
« 3
SD:
CV:
CL:
UCL:
LCL:
CLx:
A2:
°3 *
7/9/85
7/22/85
33
3l
Ji
2. f
4.3
6. o
4
8/2/85
37
32
33
/I, o
^ o?t *2
S
8/13/85
—
—
—
8/23/85
44
32
<3/
35
Co
36. o
a;
/ o
9/24/85
3&
33
31
37
3.2
/o,3
5' 7
C
10/4/85
3?
38
32
3i
3, <5
H.3
/o.i
7
10/17/85
3i
H
30
32
Ji I
H3
//, $
7
10/25/85
Ji
24
Jo
Co
3i,0
JO, 0
/->
11/22/85
fl
44
jo
3&
74
H3
/ii
/*
12/17/85
—
9/24/85
30
3?
37
35
4.1
21,3
/3.S
;
Standard Deviation
Coefficient of Variance
Control Limit
Upper Control Limit
Lower Control Limit
A2 x R =/j;j/ JJ : %0
Factors for Average = 1.023 (3 counts)
Factors for Range = 2.575 (3 counts)
Factors for Range = 0 (3 counts)
H-322
-------
WORK SHEET FOR DATA QUALITY CONTROL CHART
Strain TA98 Sample J-jjQ Activation J % 3 j
Exp. Date
Plate
Count
Average
SD
Variance
cv
Range
(1)
(2)
(3)
X
(T
O"1
r
— x 100%
X
7/9/85
—
—
—
—
—
—
—
—
7/22/85
y)
3b
40
33
1
4,5
4
8/2/85
3?
$4
/s,o
/CO (J
•>i 3
/ 1
8/13/85
—
—
—
—
—
—
—
—
8/23/85
3i
4l
4C
IS
1'.*
do J
i'f
9/24/85
S7
41
S2
Sd
4.o
/&. J
z<
i
10/4/85
J5
14
4$
6,1
7C o
/&,!
/(
10/17/85
4)
4o
37
4*
C,s>
yj.o
/?.;
10/25/85
45
SS
41
fo
7.o
410
/i.o
/j
11/22/85
a
to
$£
SI
S,l
/4, J
/c
12/17/85
a
It
41
SC
~>JS, O
->£. S
9/24/85
1-4
4i
4.1
^3
/*• 7
?
10/25/85
35
44
46
42
C.l
44. 3
/5,1
/ 3
Total n= ll
£x= /
xr= a;
SD:
CV:
1. X = IX
f n = 41.4
CL:
2. UCL- =
A
x + cl- = / /4. 7 - fit 1
A
UCL:
3. LCL- =
x - CL. =*7,4-/4, 1 --JJ.l
A
LCL:
4. R = £R
T n = /44
CL-:
5. UCL- =
d4 x r = J,£75 s - 3?, f
A2:
6. LCLp =
D3 X R = 0
V
Standard Deviation
Coefficient of Variance
Control Limit
Upper Control Limit
Lower Control Limit
A X R = / /-t,f
Factors for Average = 1.023 (3 counts
Factors for Range = 2.SIS (3 counts)
Factors for Range =0 (3 counts)
H-323
-------
WORK SHEET FOR DATA QUALITY CONTROL CHART
Strain TA98 Sample /4i 0 Activation /.j /o Si
Exp. Date
Plate
Count
Average
SD
Variance
cv
Range
(1)
(2)
(3)
X
r
(T*
r
— X 100%
X
7/9/85
_
7/22/85
4i
ft
4/
S,o
•>5
/-?, J
/->
8/2/85
44
5 /
d.t
/£
8/13/85
47
*!
4C
6,i
M
/«>./
//
fl/23/85
41
5-?
5L3
Si 1
jC
; 7
/0
9/24/e-
a
6'l
iJ
43
/o 0
10/4/85
41
£2
41
i-J
7.5
a
/4, 7
/V
10/17/85
St
$L
i 1
n
*7
3
$,o
jf
10/25/85
47
4(
C 0
4V
7.6
61
/S, 5
/4
11/22/85
$0
41
tl
41
AS
2
J, /
12/17/85
—
—
—
—
—
—
—
9/24/85
42
5-7
Cb
S4
/»&
//7
0
c? /
10/25/85
4i
42
41
45
7
* S • °>7, 5
a2:
Factors for Average = 1.023 (3 counts)
6. LCL= = D, x R = 0
K 3
V
Factors for Range = 2.575 (3 counts)
V
Factors for Range =0 (3 counts)
H-324
------- |