United States Environmental Monitoring Systems
Environmental Protection Laboratory
Agency Las Vegas NV 89114
EPA/600/4-85/058
September 1985
vvEPA
Research and Development
Guidelines for
Preparing
Environmental and
Waste Samples for
Mutagenicity (Ames)
Testing:
Interim Procedures and
Panel Meeting
Proceedings
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EPA/600/4-85/058
September 1985
GUIDELINES FOR PREPARING ENVIRONMENTAL AND WASTE SAMPLES FOR MUTAGENICITY
(AMES) TESTING* Interim Procedures and Panel Meeting Proceedings
by
ICAIR
Life Systems Incorporated
Cleveland, Ohio 44122
Contract Number 68-03-3136
Technical Monitor
Llewellyn R. Williams
Quality Assurance Division
Environmental Monitoring Systems Laboratory
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
ii
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CONTENTS
age
Figures ....................... " ..........
Tables ............................... .'.'.' vli
Acknowledgments ................... ......
1. Executive Summary ............... • ........
Introduction ........... • ............
General issues and resolutions ...............
Protocol-specific issues and resolutions ........ • • J>-
Panel meeting consensus on sample preparation protocols . • 10
2. Air Particulates , ..... ..................
Review of the literature .................. *;?
References .........................
Air particulates workgroup report .............
Protocol for the preparation of air particulates for
mutagenicity testing ................... *|j
References. . .......................
3. Drinking Water ....... ..................
Review of the literature .......... ........ °'
74
References ............... ..... .....
Drinking water workgroup report
Protocol for the preparation of drinking water for
mutagenicity testing. . . .
References
4. Environmental Waters and Wastewater ...............
Review of the literature ............... . • • •
References ..................... .**.'*
Environmental waters and wastewater workgroup report. . . . 120
Protocol for the preparation of environmental waters and
wastewater for mutagenicity testing ........... 124
References. .... ....................
5. Nonaqueous Liquid Wastes . .
Review of the literature
References
Nonaqueous liquid wastes workgroup report
Protocol for the preparation of nonaqueous liquid wastes
for mutagenicity testing
References. . .......................
iii
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6
CONTENTS (Continued)
Page
Soils and Sediments
Review of the literature. ..!!.' J"
References 162
Soils and sediments workgroup report! ! 170
Protocol for the preparation of soil and sediment samples "
for mutagenicity testing
References *v
188
7. Waste Solids .....
Review of the literature
References. .'.'.'. 19°
Waste solids workgroup report .....*.'.'!."*
Protocol for the preparation of waste solids"for
mutagenicity testing.
References .
• • 218
Appendices
A. TR-506-105B, list of participants, mutagenicity sample
preparation protocols panel meeting 219
B. TR-506-10fi oM«^ mutagenicity sample preparation protocols
222
High performance liquid chromatography protocol
227
iv
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FIGURES
Number
1 Fractionation scheme for the extraction of chemicals from
ambient air particulates (adapted from Hughes et al. 1980) . . 20
2 Isolation and f ractionation of organics from diesel exhaust
particulates (Huisingh et al. 1978) ........... ... 21
3 Acid-base extractive separation of complex mixtures (Guerin
et al. 1978) ......................... 22
4 Flow scheme for the preparation of samples from ambient air
particulates .......... ............... ^
5 Assembled high-volume sampler and shelter (Federal Register
1971) ............................. 53
6 Diagrammatic presentation of connections for sorbent cartridge
for high-volume sampler (Lentzen et al . 1978) ......... 54
7 Soxhlet extractor ....................... • 59
8 Kuderna-Danish evaporative concentrator ............. 60
9 Schematic of nonvolatile residue organics concentration
Q1
apparatus ........................... 7i
10 Details of nonvolatile residue organics concentration
00
apparatus. ......... • ......... ....... ->t-
11 Wastewater sample processing flow diagram. ... ........ 122
12 Sample information .......................
13 Sample preparation scheme for bioassay .............
14 Sample collection form ................. .... 154
15 Desiccator assay ........ ................ 156
16 Data report sheet ........................ 160
17 Chemical procedures, bioassay-directed chemical analysis .... 163
18 Fractionation scheme for soil and sediment samples ....... 182
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TABLES
Page
110
2 Origin of industrial wastewater effluents tested in the Ames
3.SS3.V *••••..
114
3 Correlation of genetic activity in Salmonella assay to
samples °r*anlcs fro» Chesapeake Bay samples and spiked
115
4 Comparison of available processing methods ....
5 Recommended volumes and storage of samples
6 STuSEp! i982Tended f°r Varl°US tyP6S °f S°lld W3Ste
192
116
130
vi
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ACKNOWLEDGMENTS
The meeting from which this document was prepared, "Mutagenicity Sample
Preparation Protocols Panel Meeting," was held July 23r25, 1984, at the Hyatt
Rickey's in Palo Alto, California. The Panel Meeting was organized and con-
ducted by ICAIR, Life Systems Incorporated. The effort was performed under
Contract Number 68-03-3136, Work Assignment 28, for the Quality Assurance Divi-
sion, Environmental Monitoring Systems Laboratory, Las Vegas, Nevada (EMSL-LV),
U.S. Environmental Protection Agency (EPA). The Panel Meeting was co-sponsored
by the U.S. Army Medical Bioengineering Research and Development Laboratory
(MBRDL).
We wish to gratefully acknowledge the administrative services of the
individuals responsible for program organization, execution, and the subsequent
preparation of these proceedings: Mr. J. Gareth Pearson and Dr. Llewellyn R.
Williams of EMSL-LV and Mr. Jeffrey S. Heaton, Mr. Jonathan P. Hellerstein,
Ms. Cynthia D. Patrick, Ms. JoAnn M. Duchene, Ms. Barbara A. Markovic, and
Mrs. Joan E. Payerchin of ICAIR.
Special appreciation is extended to the Panel Meeting Participants and the
Workgroup Leaders for the excellent quality of the presentations, the pro-
fessional manner in which technical discussions were conducted, and the quality
of the resulting protocols. Dr. V.M.S. Ramanujam, Dr. M. Wilson Tabor, Dr. Yi
Wang, Dr. Kirk Brown, Dr. Barry R. Scott, and Dr. David J. Brusick served as
Workgroup Leaders.
The efforts of all these individuals contributed to the success of the
Panel Meeting and the development of the media-specific sample preparation
protocols for short-term mutagenicity testing which appear in this document.
vii
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SECTION 1
EXECUTIVE SUMMARY
INTRODUCTION
This publication is a compilation of the review papers and sample
preparation protocols presented, discussed and revised during the meeting
entitled, "Mutagenicity Sample Preparation Protocols Panel Meeting," held July
23-25, 1984, in Palo Alto, California. The Panel Meeting was organized and
conducted to facilitate development of sample preparation protocols for
mutagenicity testing of six media: air, drinking water, nonaqueous liquid
wastes, soils and sediments, waste solids and wastewater.
Background
The available interim Ames testing procedures, under evaluation in a
multi-laboratory testing program sponsored by the U.S. Environmental
Protection Agency (EPA), do not address sample preparation for wastes and
environmental materials. Further, there are no generally accepted or standard
methods for preparing these types of samples. Both EPA's Environmental
Monitoring Systems Laboratory-Las Vegas, NV (EMSL-LV), and the U.S. Army
Medical Bioengineering Research and Development Laboratory (MBRDL) need
procedures for the preparation of wastes and environmental samples for
mutagenicity testing to ensure comparability of test results for scientific
evaluation and for potential hazardous waste site enforcement actions and
litigation.
Objectives
The overall objective of the project was to develop sample preparation
protocols for mutagenicity testing of wastes and environmental samples from
six media: air, drinking water, nonaqueous liquid wastes, soils and
sediments, waste solids and wastewater.
The following objectives were established for the Mutagenicity Sample
Preparation Protocols Panel Meeting:
1. Evaluate the adequacy and validity of the selected and reviewed
1 sample preparation protocol in each medium.
2. Prepare revised sample preparation protocols in accordance with
review comments and recommendations from the Panel Meeting
participants.
3. Present a scientific basis for any disagreements or unresolvable
issues.
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additional research to SUpport: further
^if*" sample preparation protocols.
Approach
pnnlin *• * ° p3St research °^ demonstrated experience in
'
progress reports from Workgroup Leaders at the Generll Sessions tn
""" "
" »•
^
were presented to all Panel Meeting parttclpants. A consensus a^
r
GENERAL ISSUES AND RESOLUTIONS
This section summarizes the major issues identified at the General
f fcl?e/anel Meetin8 «* the resolutions proposed by
nd Potoco0" whietha ' ""r t0,the ReVl6W PaperS« W°rk^°UP u
and Protocols which appear in subsequent sections of this report.
Media Definitions^
Sample preparation media were defined by the participants to assist
elinitio^rwe111 ^^ *** ^^^ P^tocol'for a specific Lmpl . The
mental materT'r ^ f tO enC°mp3SS the co"tinuum of wastes and environ-
To reflect the s^n ^ IT ^^-P,^^*" individual medium definitions and
developed: P protocols. The following media definitions were
aJe 20r^CUlate ^r8^ Particulates collected from the air which
are 20 microns or less in diameter.
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• Drinking Water: Water intended for human consumption.
• Environmental Waters and Wastewaters: Waters containing less than
50% suspended solids by weight, including water from lakes, ponds,
lagoons, estuaries, rivers, streams, effluents and groundwater.
• Nonaqueous Liquid Wastes: Liquids whose major component is not
water. These materials range from soluble organic liquids (e.g.,
acetone) to insoluble organic liquids (e.g., dichloromethane) and
liquids such as light to heavy oils.
• Soil and Sediments: The unconsolidated material on the earth's
surface capable of supporting plant growth and soil material de-
posited and remaining in an aquatic environment, respectively.
• Waste Solids: Waste or complex mixtures, less than 50% water,
having relative firmness, coherence of particles or persistence of
form as matter that is not liquid or gaseous at 25 C. This includes
tarry material that is sticky or viscous, such as coal tar, adhesive
waste, sludge, airborne particulates (e.g., re-entrained particles
blown from a waste site) and solids partitioned from wastewater.
Examples and further elaboration of these definitions are contained in each
Protocol.
Scope and Limitations of Protocols
Discussions to determine scope and limitations provided the following key
points, requirements and resolutions for the protocols:
• They must provide end products that meet minimal input requirements
for Ames mutagenicity testing.
• They should be quantitative to enable evaluation of any dose-
response relationships.
• They must be developed with consideration of the ultimate user
(i.e., comprehensive, stepwise procedures designed for the inexperi-
enced user).
• They will be used to determine whether samples are mutagenically
active or non-active (i.e., positive or negative types of determina-
tions) .
• They will be applied to screen large numbers of complex mixtures
within a variety of media.
• Development and standardization of the protocols will be a dynamic
process that involves establishing interim procedures that are
optimized and modified as a result of validation testing.
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Toxicity
SSriSfflw *•- ••
• AJose level of > 1 mg/plate of residue organic matter is achieved;
• The background lawn is not affected; and '
* 6™8 reVertantS are c°™t:able and comparable to control
=•
regarding the above criteria included the following: n°ernS exPressed
* ^°^slble misinterpretation by the user that testing at the
1 1 mg/plate level is required to prove non-mutagenicity.
i t0 the Mr Protoco1 because the amount of
materxal collected is often insufficient to allow dosing of > 1
mg/plate with multiple strains and replicate plates.
hrnLc . USln§ a high-performance liquid chroma-
M .- technique in lieu of the acid-base extraction procedure, the
"8 PantS iden"fled " as '
techni "8tr- Pant,S iden"fled " as - Potential sampleprepartn
technique if toxxcity is detected. The available techniques to reduce or
: aCt±Vlty **** ^^^ ""tagenic components of" mature
1. Liquid-liquid acid-base extraction f ractionation
1. Thin-layer chromatography (TLC)
3. Florosil chromatography
4. Low-pressure chromatography
5. Diction of neat material with dimethyl sulf oxide (DMSO)
o. HPLC f ractionation
activltv of^r °n."aCtive comP°"nds and potential alteration of mutagenic
activity of the acid-base extraction procedure were a maior concern. The TLf
method was not recommended because of compound loss due Jo reactiv^y of the
"oeJ; The F1H°rlSi; rd 10W-P— e chromatographic mlthLf were
to be research methods.
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The HPLC technique was viewed as the most recent technology that may be a
viable alternative to acid-base extractions. However, the following issues
were raised concerning HPLC:
Time required to run step gradient
Ability to screen large numbers of samples
Reliability and column life
Equipment down-time
Operating costs
Training level of operator
Incompatibility of certain compounds to HPLC
\
The Panel Meeting participants concluded that both the acid-base extrac-
tion and HPLC techniques should be identified in the protocols and the choice
of fractionation methods should be at the discretion of the user. A Review
and Protocol for the HPLC technique are provided in Appendix 3.
PROTOCOL-SPECIFIC ISSUES AND RESOLUTIONS
The following summaries present the major protocol-specific issues voiced
at the Panel Meeting with the resolutions achieved.
Air Particulates Protocol
The Air Protocol is restricted to sample preparation of particulate
organic material (POM) and excludes vapor phase and volatile organic com-
pounds. Mutagenicity testing of volatiles and vapor phase organics is a
research technique rather than a routine screening or monitoring method
appropriate for current needs of the EPA and the Army. The definition of "air
particulates," presented earlier, clarifies the scope of the Protocol.
The Protocol focuses on preparation of collected particulate samples,
rather than addressing detailed methods for sample collection, sampling
strategy and equipment. The available sampling techniques and equipment
(e.g., Hi-Vol sampling of ambient air) are presumably capable of providing a
sufficient quantity of POM for sample preparation via the Protocol and of
yielding quantities that meet the input requirements of the Ames mutagenicity
assay. Compositing samples from multiple Hi-Vol samplers or using scaled-up
samplers (i.e., Ultra Hi-Vol Sampler) make it possible to collect a sufficient
quantity of POM where mutagenicity bioassays, gravimetric determinations,
chemical analyses or sample archiving are planned. Generalized procedures for
particulate collection are inappropriate, since selection and implementation
of appropriate methods are source dependent.
The Soxhlet technique is recommended as the primary extraction method,
but sonication is included as an alternative. Side-by-side validation studies
for evaluation of these alternative extraction techniques are required to
determine their equivalency.
Several available options for extraction solvents are included in the
Protocol to give the investigator flexibility. These include using a single
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solvent (cyclohexane or dichloromethane) , sequential solvent extraction
(cyclohexane, then dichloromethane) or solvent mixtures (cyclohexane,
d
dichloromethane and methanol).
recommended to evaluate the need for solvent exchange
Drinking Water Protocol
Amo vA iS Umited t0 "resldue organics" that can be
absorbed on XAD resins and recovered by solvent elution techniques. The
™£MI 7 °^ i Pr?tOC°1 t0 address h±8hlv Pol"> ^nic species and highly
volatile and low molecular weight organic compounds that are not absorbed or '
recovered from the collection apparatus is emphasized. The Protocol is
applicable for all types of finished drinking water. The type of disinfection
treatment (if any) Js not considered to be a critical issue. A glass wool
pref liter, a Celitev column and a bacterial filter are incorporated when
waters contain more than 5% solids, are not disinfected or contain more than
20 parts/million (ppm) total organic carbon.
Detailed methods for cleaning the XAD resins are presented to ensure that
a uniform approach is implemented for determinations of resin purity and to
eliminate potential for contamination. Other quality controls include blanks,
spiking of samples, controls, duplicates and frequency of sampling.
Additional research is needed to evaluate using additional columns such
as cation and anion exchange resins to recover highly polar or ionic species
from dilute aqueous solutions. Recovery studies using a variety of organic
compounds with a wide range of polarities and solubilities are recommended to
determine the efficiency of this system. Radiolabeled compounds and a variety
of surrogate chemicals should be used to evaluate irreversible binding to
resins and to quantify compound loss in the collection apparatus.
Environmental Waters and Wastewater Protocol
This medium is multi-phasic and covers a wide range of constituents,
including aqueous and nonaqueous liquids and dissolved and suspended solids
The Protocol is limited to solvent-extractable organic compounds; however, not
all compounds will be recoverable and/or stable under the Protocol's methods
An overall scheme was developed and incorporated in the Protocol to link the
variety of components of this medium to the other Protocols.
(a) Registered trademark.
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Each discrete phase of this multi-phase medium is collected, quantified,
separated and concentrated or extracted during sample preparation. Gravity
separation (24-hr duration) of the sample into three distinct phases initiates
the process. Standard methods for determinating percent suspended solids in
the aqueous liquids are included in the Protocol. The Protocol for Drinking
Water is used to prepare aqueous liquids with less than 5% solids. Aqueous
liquids with more than 5% solids are prepared by one of the following methods:
(1) liquid-liquid extraction, concentration and solvent exchange; (2) separa-
tion by filtration or centrification, with the liquids being processed accord-
ing to the Drinking Water Protocol and the solids according to the Waste
Solids Protocol. The solid sediments from the initial 24-hr gravity separa-
tion are processed according to the Protocol for Waste, Solids. Nonaqueous
liquid wastes partitioned from the samples are processed via that Protocol.
Nonaqueous Liquid Wastes Protocol
Nonaqueous liquid wastes were defined to include compounds that range
from water-soluble organic liquids to immiscible oils. Only a limited amount
of data are available on the applicability of this Protocol to compounds other
than oils or petroleum products. This medium differs from the other media
because materials are often concentrated. The Protocol incorporates dilution
steps in addition to the concentration techniques used in the other media
Protocols. This Protocol is unique because of the opportunity to test neat
material or neat material diluted with DMSO without losing polar compounds.
This medium enables analyses with complete samples rather than with compounds
collected on an absorbent or extracted with a solvent. Testing using neat
samples or DMSO suspensions is recommended as an initial test.
Procedures for solvent extraction are also included in this Protocol as a
second step. Dichloromethane is recommended as the choice of solvent rather
than diethyl ether for several reasons. Dichloromethane promotes uniformity
with the other Protocols' solvent selection, is more readily obtainable in a
high purity form and reduces potential safety problems. Identification of the
optimum solvent(s) remains a research question that should be addressed by
extraction efficiency studies comparing various solvents, mixtures or
sequences.
No specific time requirement for storage is specified due to the lack of
available scientific data on degradation; however, it is recommended that the
neat material be tested as soon as possible.
No adjustment of pH is recommended for the neat sample. In cases where
fractionation is required, extraction of bases with hydrochloric acid and
subsequent separation of the acid and neutral fractions are recommended to
avoid destruction of mutagens. The acid-base fractionation procedure is
identical to that used in the Soils and Sediments Protocol and the Waste
Solids Protocol. The Workgroup made specific recommendations not to perform
additional fractionation if toxicity is observed in the DMSO acid, base or
neutral fractions. The acid-base fractionation procedures are not suitable
for low concentrations of highly polar mutagens. The HPLC technique is an
appropriate alternative to the acid-base extraction method.
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for
°f
-.pounds
.
for
control and a
'"ting
Soils and Sediments Protocol
information on specific soil cont-^f f ? fPPr°Priate in cases where
appropriateness of using rsingleL^t8 ver± "'6 " available'
for preparation of soils' remains a reLarch issue?
°r
medthod rcommended because °
la otdl8cretiootn
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Optimum storage conditions for soils and sediments remain presently
undefined. There is an absence of quantitative data on the stability of
organic pollutants in soil and sediments under a variety of storage condi-
tions. Experiments on degradation of spiked compounds under different storage
temperatures, moisture content, storage times and soil or sediment types are
recommended to determine optimum conditions. Autoclaving is discussed as a
technique that may improve soil stability, but resulting alteration of
mutagenic activity needs to be addressed by further research.
t
Waste Solids Protocol
The Protocol's scope is limited to organic compqnents of waste solids
that are solvent-extractable and remain stable through the sample preparation
procedure. This Protocol is based on well established analytical chemical
methods that have been used extensively to determine compositions of complex
organic mixtures and environmental samples. However, only limited data are
available to demonstrate the efficacy of such methods to prepare waste solids'
samples for mutagenicity assays. Vapor phase organics and inorganic com-
ponents of waste solids are intentionally not addressed in the Protocol.
Waste solids are defined as heterogeneous materials that range from
sticky, viscous or tarry material to dry solid particulates. Special tech-
niques for treatment of oily, gummy and adhesive materials, (e.g., addition of
anhydrous sodium sulfate or silica gel) are specified in the Protocol. The
gravity phase separation procedure (24 hr at 4 C) developed by the Environ-
mental Waters and Wastewater Workgroup is incorporated by reference to address
removal of liquids from waste solids samples.
The Waste Solids Protocol applies to waste solids samples partitioned
from aqueous or non-aqueous liquids and potentially from ambient air or
gaseous media. Freezing samples or adding dry ice prior to grinding for
particle size reduction are considered comparable alternative pretreatment
procedures. Reduction of waste solids to a maximum particle size of 2 mm or
less is recommended to enhance the extraction procedure.
The extraction procedure is a key issue. The Soxhlet technique is
selected as the primary method with use of a blender as an acceptable
alternative. Additional research is recommended to evaluate efficiencies and
equivalency. The addition of surfactants or other compounds to improve
extraction efficiency is not recommended except in cases where supporting
scientific data are indicative. Dichloromethane is the recommended solvent.
Use of DMSO is limited due to interferences with chemical analyses and
reduction in extraction efficiency. Extraction should be conducted at 4 C to
reduce degradation of complex mixtures. The efficiency of specific solvents,
sequences or mixtures for the extraction of waste solids remains a research
issue.
The K-D apparatus is the primary method for solvent volume reduction;
however, the rotary evaporator may be used to concentrate extracts when the
loss of moderately volatile chemicals is not of concern. The solvent DMSO is
used to redissolve an aliquot of the concentrated crude extract from the K-D
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°ther
alt*/- W?rk8rouP acknowled8" that acid-base extraction can potentially
" ^8 recommended f°^ all samples and at -20 C
less forcr pes an at - C or
f rs s^s: are mb-
storage of raw samples, crude extracts and processed extracts.
PANEL MEETING CONSENSUS ON SAMPLE PREPARATION PROTOCOLS
Air Particulates
Air P-L\^^
nHoeerh *? f*^'-*^ of ^ Particul^tL for mutagL ^ity
« J H°Te ', Part±^Pants agreed that there is not a sufficient data
Protocl H ^ ?f±0n °f the etlulval^y of alternatives provided in this
Protocol, such as (1) selection of solvent(s) for extraction (±.e single
: - - -
solvent selection, volume reduction and solvent exchange.
10
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Drinking Water
The Panel Meeting participants were in unanimous agreement that the
Drinking Water Protocol is a well established method with a sufficient founda-
tion of data that demonstrate the adequacy of its application to isolating
residue organics from drinking water for mutagenicity testing. This consensus
to recommend the Drinking Water Protocol was made with cognizance of the
inherent limitation that highly polar and ionic species or highly volatile low
molecular weight organics may not be recovered. The Panel Meeting
participants recommended that the Protocol be subjected to validation studies
by various laboratories. Additional research on the method was not considered
necessary. ^
Environmental Waters and Wastewater
The Panel Meeting participants were in unanimous agreement that the
Environmental Waters and Wastewater Protocol is a synthesized protocol that
assembles portions of a number of established methods. There is a good
indication that this combination of components will work together; however, a
comprehensive data base demonstrating the efficacy of the hybridized combina-
tion of methods needs to be generated and evaluated.
Nonaqueous Liquid Wastes
There was unanimous agreement among Panel Meeting participants that the
Nonaqueous Liquid Wastes Protocol is a synthesized protocol that assembles
portions of a number of proven methods. There is a good indication that the
various hybridized components will be applicable to a range of nonaqueous
liquids; however, this is based on available data from testing of discrete
types of oily compounds. Further studies were recommended to evaluate applica-
bility to the variety of nonaqueous liquids and to determine the optimum
solvent for separation or fractionation of these compounds.
Soils and Sediments
There was consensus among the Panel Meeting participants that the Soils
and Sediments Protocol is a synthesized protocol that assembles a combination
of well-established techniques. However, the data base demonstrating adequacy
of components in this combination is not extensive. There is a good
indication that the Protocol is applicable for preparation of both soils and
sediments for mutagenicity testing. One dissenting participant expressed
concern with the use of water-insoluble solvents for extraction of soils and
sediments. The recovery efficiencies of methods using sodium sulfate or
silica gel as drying agents and extraction with dichloromethane are not well
documented and were the basis of the disagreement.
Waste Solids
There was unanimous agreement among Panel Meeting participants that the
Waste Solids Protocol is a synthesized protocol that integrates a number of
well-established methods and offers a number of alternative techniques. There
is a good indication that these methods are applicable for preparation of
11
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additional research "I^aring^xtracti101*7 5eStlng' but there is need for
blender versus sonicator), choice nf «!!?, t6* /qUM f±*e'» s°xhlet versus
single solvent, solvent series etcS «H /"" °u DMS° f°r Crude extract,
sodium sulfate). ser^s, etc.) and drying techniques (silica gel or
12
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SECTION 2
AIR PARTICULATES
REVIEW OF THE LITERATURE
Introduction
The presence of chemicals in the air is a fact of life in a modern
society, since an increasing number of chemicals have been released in large
amounts into the ambient environment. The chemical industry, automobile
exhaust and fossil fuel burning contribute many organics and metals_to the
air. Thus, the urban atmosphere contains a complex mixture of chemicals, many
of which are toxic, mutagenic and carcinogenic. Also, it is not unreasonable
to expect that products from reactions of these chemicals with N02 and SO,,, by
photochemical or other processes, will be observed in the atmosphere (Calvert
and Pitts 1966, Gould 1976, Hirayama et al. 1983, Leighton 1961, Lindskog
1983, Pellizzari 1978, Pitts 1983).
Many known mutagens and carcinogens have been detected in the ambient
atmosphere (Broddin et al. 1980, Fishbein 1976, Singh et al. 1983, ««ers et
al 1978). The atmosphere also contains many compounds whose mutagenic effect
is'unknown. Short-term mutagenicity test systems with a certain predictive
value for carcinogenicity are now available and can be used to assess the
mutagenicity of extracts of complex environmental mixtures (Harnden 1978,
Waters et al. 1978). Shortly after the development by Ames j19^' ^75), of a
sensitive microbiological assay for mutagenicity, Pitts et al. (1977) ^ported
preliminary data on mutagenic activity in the organic fraction of ambient air
borne particulates using the Ames assay system. In later years, many other
studies were undertaken using predominantly the microbial assay system
(Brusick and Young 1982, Chrisp and Fisher 1980, Drummond et al. 1982, Hughes
et al. 1980, Sexton et al. 1981). Mutagenic activity was observed in ambient
particulate samples from industrial or urban areas (Alfheim et al. 1983a,
Commoner et al. 1978, Dehnen et al. 1977, 1981, Talcott and Wei 1977, Tokiwa
et al. 1977, 1980, Hoffmann et al. 1980, Hughes et al. 1980, Krishna et al.
1983a, b, Moller and Alfheim 1980, Wang et al. 1980, Wei et al. 1980 , and in
particulate matter from special sources such as automobile and diesel exhausts
(Huisingh et al. 1978, Rohan and Claxton 1983, Lewtas 1983, Nakagawa et al.
1983, Ohnishi et al. 1980, Rannug 1983, Rappoport et al. 1980, Schuetzle 1983,
Stenberg et al. 1983a, b, Wang et al. 1978, Wei et al. 1980), fly ash from
coal and other combustion processes (Alfheim et al. 1983a, b, 1984, Kubitschek
and Venta 1979, Kubitschek and Williams 1980, Lofroth 1978, Moller and Alfheim
1983), coal-liquefaction products and crude and shale oil samples (Alfheim et
13
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and r'g ' ^ " a1' 1978' Thl11" « <"• "", Vouk
procedures into a useful and cohesive document.
Sources and Types of Air Pollutants
?hemlcal and biol°8ical characterization of Atmospheric pollutants is
organic matter (POM) (NAS 1972, NRC 1979), it is important that collection
st^3 S an analy?lcal techniques encompass the entire spectrum OJ sub-
stances (Hughes et al. 1980, Krishna et al. 1983a, b) .
Dollnt«nf. are =lasslf±ed as primary and secondary. Primary air
r^of t f "^T1 CdUStS' ^^tation, etc.) or anthropogenic in origin
(smoke stack vehicle emissions, etc.) (Fishbein 1976, HugLs et al. 1980)
reactions fS^ ^ ? ^rate* from primary pollutant's via atmospher^
reactions (photochemical, reactions with NO , SO , 0 , etc.).
£ £, J
?^TUn^ °f JaP°r-Phase organics emitted anthropogenically in the
States has been estimated to be 1.9 x 1013 g/yr. The levels of
larticul^ °rganjCS a5e ««;«lly 10 to over 100 times greater than those of
particulate organics (Duce 1978, Hughes et al. 1980).
The relative distribution of air pollutants between POM and VPO is
dependent upon their vapor pressure, polarity and ambient air-temperature
VPO dllTl' SUCCT fUl ChemiCal 3nd biol°^*l analysis of the POM and
VPO depends upon a number of important steps, starting with a collection
and/or pre-concentration technique (Hughes et al. 1980).
Toxic and Genotoxic Chemicals Identified in Air
The insult to human health and ecosystems from airborne chemicals is due
to several families of genotoxicants which include: aldehydes- aliphatic and
aromatic amines; alkenes; asbestos; a.aarenes (mono-, di- and
, c;
i; FT**?* halogenated alkanes and alkenes; long-chain
_ acids, alcohols, hydrocarbons and esters; metals and their com-
pounds; nitrosamines; NO; nitroarenes; oxidants (0,, PAN); polycyclic aromatic
°C
' - « ros an ng
, Cleland 1981, Cupitt 1980, Fitzpatrick et al. 1983, Gibson 1982 1983
iei98 * KT' ^^T 6t a1' 1983* H°Wle 3nd K°eSte;s 1983' ^bbekus and
i 1983, Kolber et al. 1983, Kohan and Claxton 1983, Levins 1982
14
-------
The primary sources of PAH in the United States are thought to be fuel
combustion for power and heat generation and the open burning of refuse (NAS
1972). Estimates of the annual emissions of benzo(a)pyrene are up to 500 tons
per year for heat and power generation and 600 tons per year for refuse burn-
ing. Of the latter figure, 140 tons per year are from forest and agricultural
fires. Forest fires are also a major source of total suspended particles
(TSP); estimates range from a low of 0.5 million tons per year to a high of
54 million tons per year (Ward et al. 1976).
Sources of airborne nitroarenes, which are direct-acting bacterial
mutagens (in the Ames S. typhimurium assay) (1-nitropyrene, dinitropyrenes,
6-nitrobenzo(a)pyrene, etc.), include diesel-powered and gasoline-powered
vehicles, woodburning fireplaces, residential heaters and coal-fired
industrial furnaces (Gibson 1982, Kohan and Claxton 1983, Nakagawa et al.
1983, Pitts 1983, Zweidinger 1982). Presumably, most combustion sources emit
these nitroarenes, which are thought to be formed by the reaction of N0x gas
with PAH.
Collection Techniques
The accuracy and precision of a collection and/or pre-concentration
technique are important, since the materials collected must be representative
of the actual composition of the vapor-phase and particulate organic matter
occurring in the atmosphere (Hughes et al. 1980, Pellizzari 1978).
Collection of Particulate Organic Matter (POM) —
The collection of POM is generally done by using impactor systems,
electrostatic precipitators and filtration samplers. The high-volume (Hi-Vol)
sampler (which can sample approximately 2,000 m of air in a 24-h period) is
used routinely to collect aerosol, with nearly quantitative trapping effi-
ciency for particles less than 20 ym in diameter (Federal Register 1971).
Glass-fiber filters are commonly used for collection. A more recent method of
sampling particles from very large volumes of air involves the use of the
Massive Air Volume Sampler (MAVS) designed by Henry and Mitchell (1978), which
samples over 2,000 m of air in a 24-h period and employs impactors and an
electrostatic precipitator to provide aerosol sized into three fractions
(<1.7, 1.7-3.5 and >3.5 ym diameter) (Hughes et al. 1980, Kolber et al. 1983).
Electrostatic precipitation has the following inherent deficiencies: (1)
The collection efficiency is dependent on a number of variables, one of which
is flow rate, and sampling at the flow rate desired to collect large quan-
tities may result in poor collection efficiency of particles, especially those
of 1 ym. This is particularly significant in mutagen bioassay, since the
small particles have been found to have a higher content of mutagenic
material. (2) The sample is spread out over many square feet of metal col-
lector plates, which makes quantitative retrieval difficult and artifacts due
to handling more likely. (3) Particulate components may be susceptible to
alteration by the electrostatic field.
An ultrahigh-volume sampler for the multiple filter collection of respir-
able particulate matter has been described by Fitz et al. (1983). These
authors recommend the use of this ultrahigh-volume sampler when mutagen
15
-------
de^1^ ^^±cal analyses of the organic extract of airborne
?* T deSlrable' Th±s "Itrahigh-volume sampler is also
recommended when short time collection intervals are desirable in order
to prevent evaporation or chemical reaction of organic compounds
^ AlfhfV^ Linfkog (1984) have recently compared different sampling
various ' f±lllr f electros « compared to those from ordinary
tJon nf ? flUers> represented filter artifacts or a more pronounced degrada-
tion of mutagenic compounds on the noncoated glass fiber filters.
°f fijtratlon Artifacts has also been addressed by Daisey et
aco • u authors comPared fiberglass and Teflon-impregnated fiber-
£ ^^ J reSP6Ct t0 ^ bacterial "utagenicity in the Ames test of
extractable organic matter. Significant differences in mutagenicity between
Sri- r° ^^er/yPeVere observed- I" addition, the responses differed among
bacterial strains For example, with S. typhimurim TA 98, mutagenicity was
greater for the polar organic fraction from the fiberglass filters; with S.
typhimurium TA100, the polar organic fraction from the Teflon-coated
fiberglass filters was greater than for the fiberglass filter fraction.
Fitz et al. (1983) have also studied the potential for the formation of
artifacts during filtration sampling. Their experiments indicate that the
bacterial mutagenicity as determined by the Ames bioassay did not depend
significantly on the filter medium. These authors also considered the
question of filter precleaning. The lack of precleaning with the extraction
solvents may lead to a filter artifact which is not due to the filter type
itself, but to contaminants.
Collection of Vapor-Phase Organics (VPO) —
The collection methods now available for vapor-phase organics include
xh trap Or8anlc vaP°rs on sorbent surfaces (Pellizzari,
™t P i ;' C°nfrSf °r freeze Vap°rs in erogenic traps (Grimsrud
and Rasmussen 1975, Tyson 1975) or confine the pollutants in evacuated stain-
less steel bags or canisters (Hughes et al. 1980). The presence of water
poses a major problem in sampling VPOs. The relative abundance of water in
the atmosphere is high (as much as 100-fold compared to the organic species of
interest), and the collection and analysis devices (such as gas chromatography
columns, mass spectrometers, etc.) do not tolerate large quantities of water.
After careful evaluation of several sorbents for air sampling of VPOs,
^llZl "workers (Bursey et al. 1977, Pellizzari 1974, 1975, 1977a, b,
1978) have recommended the following primary criteria to choose the right
sorbent (Hughes et al. 1980): (1) ability to discriminate against water and
preferentially concentrate the VPOs of interest, (2) minimal decomposition or
16
-------
polymerization of the sample constituents during collection and recovery,
(3) high breakthrough volumes, (4) quantitative collection efficiency and
recovery of trapped vapors and (5) collection systems that do not contribute
to in situ formation of artifacts.
Activated carbon or charcoal has been used as a sorbent material in some
VPO studies (Eppig et al. 1982, Grob and Grob 1971). However, the recovery of
the collected VPOs from charcoal is usually incomplete, and in some cases,
charcoal catalyzed reactions between individual organics present in the com-
plex mixture (Miuere and Dietrich 1973, White et al. 1970). In the cryogenic
(freeze-out) collection technique (generally employed for C2-Cg hydrocarbons),
the very volatile VPOs are generally recovered from the frozen sample by
distillation. The presence of water continues to pose technical limitations,
and the VPOs in the cryogenic trap are usually separated from water vapor
through a sequential increase in temperature before analysis.
Recent literature highly recommends the use of the Tenax-GC adsorbent (a
porous polymer of 2,6-diphenyl-p-phenylene oxide) collection technique for the
G -C volatility range of VPOs (Adams et al. 1977, Bursey et al. 1977, Harris
ef al5. 1982b, Kaiser 1974, Lutz 1982, Pellizzari 1974, 1975, 1977a, b, 1978,
Walling et al. 1982).
A recent report by Pellizzari et al. (1983) describes the preparation of
61 different polyimide sorbents that have the potential of either replacing or
complementing Tenax-GC. According to this report, four polyimide sorbents,
PI-109, PI-115, PI-119 and PI-149, are selected for further studies on
collection desorption efficiencies, artifact formation, shelf life, humidity
and the effects of inorganic species such as ozone, nitrogen oxides and sulfur
oxides.
The use of several other resins such as XAD-2, XAD-7, chromosorb 100
series, Porapak, Florisil, Ambersorb-340, Ambersorb-347 and Ambersorb-348 has
also been recommended for the sampling of VPOs in air (Harris et al. 1982a,
Lutz 1982). Florisil Is comparable to XAD-2 and Tenax-GC in terms of its
volumetric capacity for collection of the alkanes, alcohols and chlorinated
aromatics. Florisil shows very much higher (about 10,000 times) capacity for
collection of volatile chlorinated aliphatic compounds such as dichloroethanes
and dichloropropanes. Ambersorb XE-340, Ambersorb XE-347 and Amberlite XAD-7
have a substantially greater capacity for collection of low molecular weight
alcohols than do XAD-2 and Tenax-GC.
Preparation of Extracts for Bioassays
Introduction—
As Guerin et al. (1978) point out, a major consideration in the muta-
genicity testing of complex chemical mixtures is that such mixtures contain
chemicals of a wide variety of classes and an equally wide range of concen-
trations that are potentially capable of causing additive, antagonistic or
synergistic responses in test organisms or at specific receptor sites. Such
interactions may be a function of the combined dose of toxicants or of the
inherent genetic susceptibility of a particular target organism. The prepara-
tion of complex mixtures for in vitro bioassay (e.g., Ames test) is com-
17
-------
t^the'test ^ ^ f° ^Ct°rs: (1> the relevance of the material applied
to the test system and (2) the compatibility of the material to the test
t'esTsv tU6rf f 'I' I9?0' ChemiCal "rele—" IB achieved when the
test system is dosed with a material whose chemical composition mimics that
which reaches the natural point of impact. Difficulties with "compatibility"
which S T°UnterKd fen the -aterlal being bioassayed contains constituents
T^ f «6rK the teSt Or8anlsm8' ability to respond to the effect of
interest High concentrations of mildly toxic constituents or small quanti-
ties of highly toxic constituents can sometimes mask the more subtle effect of
mutagenic constituents. Any steps taken to remove toxic constituents in order
to make the material compatible with the test system necessarily involve a
change in the physical or chemical nature of the test.material. Thus, the
objective of selecting the best sample preparation protocol is to choose a
procedure which prepares materials in a form suitable for biotesting with a
minimal or at least interpretable impact on the relevance of the test
material.
Solvent Extraction of POM Samples—
rh.m-STle ?"paration is a costlv ^ature in mutagenicity testing. When
chemical purification is not the goal, samples of POM can be extracted with
organic solvents and the crude extracts tested for mutagenicity without
chemical fractionation. Fractionation is only required if the crude extract
proves to be too toxic for direct testing. Thus, a stepwise approach to
sample prefractionation is the most efficient.
Direct Extraction-
Direct extraction of air particulates without fractionation is highly
recommended. In a recent study by Krishna et al. (1983a), organic materials
were extracted from airborne particles by shaking with different solvent
systems (without fractionation), including acetone, benzene, cyclohexane,
methylene dichloride, methanol, a mixture of acetone and methylene dichloride
and a combination of benzene, cyclohexane and methanol. The solvent-extracted
materials were tested for mutagenic activity with the Ames Salmonella/micro-
somal assay system. Acetone- and cyclohexane-extracted materials gave the
highest and lowest mutagenic activities, respectively. In addition, these
authors tried sequential extraction with acetone followed by methylene di-
chloride, which gave a better mutagenic response than acetone alone or acetone
in combination with methylene dichloride.
Even though acetone has been used widely in different laboratories for
air particulate extraction, it is not a highly recommended solvent due to its
reactivity with other chemicals and unavailability in very pure form (Lentzen
et al. 1978, Brusick and Young 1981).
Other solvent systems, such as cyclohexane (Alfheim and Moller 1979)
benzene-hexane-isopropanol mixture (Commoner et al. 1978), cyclohexane,
methylene chloride and acetone serially (Daisey et al. 1979), methanol (Dehnen
et ai. 1977), methanol-benzene-methylene chloride mixture (Pitts et al. 1977
?oao ?er 1980)' acetone (Talcott and Wei 1977) and benzene (Fukino et al.
1982, Teranishi et al. 1978), have also been used for the extraction of
mutagenic material from ambient air particulates. Among these solvents,
18
-------
dichloromethane is the best choice for air particulate extraction, due to its
intermediate polarity.
Among such different modes of extraction as shaking, Soxhlet extraction
and sonication, either sonication or Soxhlet extraction seems to be the mode
of choice for the extraction of mutagens from airborne particulates (Hughes et
al. 1980, Krishna et al. 1983b, Alfheim and Lindskog 1984).
Separation and Identification of Chemicals for Biological Testing
Separation—
\
Chemical fractionation—As mentioned earlier, fractionation is required
if the crude extract proves to be too toxic for direct mutagenicity testing.
Fractionation is also needed for chemical purification and identification by
chromatographic (gas and liquid) techniques and mass spectrometry.
The partition scheme (Figure 1) proposed by Pellizzari et al. (1978) is
one of the methods of fractionation of ambient air particulates. This scheme
results in fractionating air particulates into six chemical classes: acids,
bases, polycyclic aromatic hydrocarbons, polar neutrals, nonpolar neutrals and
insolubles. This fractionation scheme essentially involves the sonication of
air particulates with cyclohexane to separate solids (containing polar
organics and inorganics) and the cyclohexane layer containing nonpolar
organics. The solids and the cyclohexane layer undergo further fractionation
through a solvent partition scheme (methanol, methylene dichloride and aqueous
acids for solids; aqueous acids, cyclohexane and aqueous methanol for nonpolar
organics in the cyclohexane layer) to produce six fractions (Pellizzari et al.
1978).
This partition scheme has been validated for its chemical efficacy by
using chemicals representative of individual chemical classes (Pellizzari et
al. 1978). Using this fractionation scheme, the various chemicals used were
recovered with a high percentage efficiency.
As a further check on the procedure, thin-layer chromatography (TLC)
scans were conducted on each fraction to ascertain the extent, if any, of
spillover of one compound into other fraction(s). No such spillover was
detected by these authors. The first sample that was examined very success-
fully using this fractionation scheme was collected at South Charleston, West
Virginia, during August, 1977 (Pellizzari et al. 1978).
Several other fractionation schemes have also been developed, including
(1) isolation and fractionation of organics from diesel exhaust particulates
(Figure 2) (Huisingh et al. 1978) and (2) acid-base extractive separation of
complex shale oil fractions (Figure 3) (Guerin et al. 1978).
Separation by chromatographic techniques—
Column chromatography: This is the most widely used separation method
for POM. A large variety of adsorbents, such as alumina, silica gel,
Florisil, cellulose acetate and gel materials, have been used for separation.
Adsorbents with uniform particle size, 60-80, 80-100 and 100-120 mesh, and
19
-------
Particulate
1. Sonicate IN C.H,.
2. Filter ~ 6 12
Res
1
Inorganic
Residue
idue Filt
1. Sonicate in
MeOH
2. Filter
rate
Combine Filtrates
1 . Remove Solvent
2. Redissolve in CH Cl
3. Acid Wash Sequence (a)
1
Aqueous
(Discard)
Aqueous
1. Basify to pH 10
2. Extract 3x with CH.CL.
1. Base Wash Sequence
' '
I
Aqueous
1. Remove Solvent
2. Weigh
I Organic Bases {
1. Acidify to pH 3
2. Extract 3x with
CH2C12
1. Remove Solvent
2. Weigh
Aqueous
(Discard)
I Organic Acids I
Insolubles
C6«12
1. Wash 3x with
MeN00
C6H12
MeNO,
1. Remove Solvent
2. Weigh
1. Remove Solvent
2. Weigh
Nonpolar
Neutrals
I PNA*S1
1. Remove Solvent
2. Redissolve in C.H,0
3. Filter 6 12
C6«!2
1. Wash with MeOH/H.O
MeOH/HO
1. Freeze Dry
2. Weigh
Polar Neutrals
(a) Acid wash sequence 2x with 10% H.SO., Ix with 20% H.SO,.
/ 4 24
(b) Base wash sequence 3x with IN NaOH.
Figure 1. Fractionation scheme for the extraction of chemicals from
ambient air particulates (adapted from Hughes et al. 1980).
20
-------
Filters
f
DCM Extract
1. Evaporate and Weigh Residue
2. Redissolve in Ether
Ether Solution with
Some Insolubles
1 Extract with Base
\
Aqueous Phase Ether
11. Acidity
2. Extract Ether __^
*
Acid Fraction fn. j\
(ACD) (Discard)
i-u-8j-u-y3% r™
1 1 • 9 • OR « S • 1 S j£
ii z uo 3 Aqueous Phase
11. Add Ba
2. Extrac
f
Basic Fraction
BAS
1-0-03-0-03
11-0-09-0-05
Soxhlet
Extraction
CH2C12 (DCM)
Filters
j Soxhlet
I Extraction CH
ACN Extract
i f
Solution
Ether Insolubles
Extract H PO, (INT)
1-0-12-0-008
11-1-18-0-700
f
Ether- Solutions
se Neutrals (NUT)
t 1-53-38-51-81
Aqueous Phase 11- 19-27- 17-30
(Discard) i
Figure 2. Isolation and fractionation of organics from diesel exhaust
particulates (Huisingh et al. 1978).
-------
Ni
NJ
Sample
Organic
Basic
And
Neutral
AQ
IN HC1
pH 11
Ether
pH 9
Bases
ORG
Bases
6"^xane
Ether (or MeCl.)
IN NaOH
Aqueous
NaOH
Insol.
Acidic
ORG
Neutral
Florisil
Column
AQ
AQ
pH 6.1
Ether ORG
WA
pH 1.0
Ether ORG
Weak
Acids
«t 0
SA
Strong
Acids
Strong
Acids
Hexane/Benzene
8/1
Benzene/Ether
4/1
Methanol
Figure 3. Acid-base extractive separation of complex mixtures (Guerin et al. 1978).
-------
column diameter:length ratios of at least 1.25 are suggested. The laboratory
temperature should be kept reasonably constant, and the solvent used for
elution should be of highest purity. Maximal separation can be achieved by
slowly and evenly increasing the polarity of the eluting solvents. The
hydrocarbons are eluted from the column in the following order: Aliphatics,
olefins, benzene derivatives, naphthalene derivatives, dibenzofuran fraction,
anthracene fraction, chrysene fraction, benzopyrene fraction and coronene
fraction (Sawicki et al. 1970).
Thin-layer chromatography: This technique is quick, inexpensive and
reasonably reproducible and is one of the better methods for the separation of
isomeric compounds. With the availability of in situ, scanning techniques
(thin-layer scanner), this method has been used for quantification of POM with
reasonably good accuracy. A number of materials have been used as sorbent
materials for the plates, including silica (Liberti et al. 1975, Candeli et
al. 1975, Hunter 1975, Dong et al. 1976); alumina (Woidich et al. 1977,
Sawicki et al. 1970a, b); cellulose (Sawicki et al. 1967); cellulose acetate '
(Schultz et al. 1973, Tomingas et al. 1977, Harrison and Powell 1975, Woidich
et al. 1977) and polyamide (Bories 1977). A number of modifications, includ-
ing silanization of silica gel (Brocco et al. 1973) and channel thin-layer
chromatography (Zoccolillo and Liberti 1976), have been proposed. In the
latter procedure, the components which need to be fractionated on a thin-layer
plate are made to flow into narrow development channels in order to prevent
the spreading of the chrotnatographic spots.
Both PAH and polynuclear aza-heterocyclic compounds have been separated
by the TLC method. A large number of variations of the TLC procedure, such as
variation of developing solvents, use of a mixture of sorbents and development
in one or two dimensions, have been used to affect better resolution of the
components. The effect of solvent variations on cellulose acetate TLC has
been discussed in detail by Woidich et al. (1977). Although alumina-cellulose
(2:1) has been used as a composite adsorbent, the best resolution is obtained
when two-dimensional TLC on a composite plate of aluminum oxide-40% acetylated
cellulose (2:1) is used (Chatot et al. 1972a,b, Pierce and Katz 1975).
A large number of aza-heterocyclic compounds (acridines and quinolines)
found in urban airborne particulates and in air pollution source effluents
have been separated by TLC procedure. The basic fraction from a coal-tar-
pitch sample was subjected to a two-dimensional TLC separation on silica
gel-cellulose (2:1) with pentane-ether (9:1, v/v) and dimethyl formamide-
water (35:15, v/v) solvent systems (Sawicki et al. 1965).
Two-dimensional TLC separation has also been successfully used during the
quantification of acridine, benz(c)acridine, 7Hbenz(de)anthracen-7-one and
phenalen-1-one in airborne particulate samples (Sawicki et al. 1966).
There are several disadvantages with the TLC procedure. Mass spec-
trometry and liquid chromatography, when applied as additional analytical
tools for identification of POM, have demonstrated that the individual spots
or bands are sometimes mixtures of two or more compounds. Also, the TLC
procedure cannot be used when separation of a large number of compounds is
23
-------
the
points demand the
.97* Lao et lo ..SL-J.S'fi'i? " -
et
1974), carbowax-20 polymer (Hill et al 1977-> nv i rr j v^ertscn et
24
-------
compared several liquid phases and found that a 50-m SE-52 column was most ef-
fective for POM separation and had a theoretical plate value of 40,000.
Investigations by Bjorseth (1978) have demonstrated that capillary columns
using SE-54 as the stationary phase have excellent separation efficiency, low
column bleed and long-term stability. Both PAH and aza-arenes have been
separated with this packing material (Bjorseth 1978). A 40-m capillary column
with Versatnid-900 as the stationary phase is very effective for the separation
of aza-arenes (theoretical plate value of 60,000) (Brocco et al. 1973).
High performance liquid chromatography (HPLC): The ability of HPLC to
separate POM compounds not normally accomplished by gas chromatography (GC)
indicates the great potential of this method for POM analysis. In addition,
HPLC offers many advantages over GLC: it operates at much lower temperatures,
fraction collection is simple and convenient, the capacity to accept sample is
high and fluorescence spectroscopic detection demonstrates high sensitivity
and selectivity for POM compounds.
In HPLC, the mobile phase modifier is the most powerful variable for
changing retention times. The choice of mobile phase is dictated by the mode
of separation accomplished by the sorbent. In the normal mode, hydrocarbon
solvents are generally used as the mobile phase. Any solvent that dissolves
POM in fair amounts, is miscible with water and is transparent at UV absorp-
tion wavelengths (for UV or fluorescence detector) can be used as a modifier
in the reverse phase-HPLC.
It should be mentioned that solvent programming has proven to be a
powerful tool in separating the compounds from each other Several research-
ers have taken advantage of increased temperature (70 to 80 C) of the column
to obtain a similar objective. At higher temperature, reduced viscosity of
the mobile phase increases column efficiency and decreases relative retention
times. The sample capacity is also increased at higher temperatures, allowing
larger amounts of sample injection. The most versatile columns for the
analysis of PAHs in POM operate on the principle of reversed-phase mechanism.
The C-18 reversed-phase column has been applied for the separation of both PAH
(Dong 1976, Flessel et al. 1981, 1984) and aza-arenes (Dong et al. 1977).
Normal-phase HPLC columns have also been used for separating extracts from
diesel exhaust particles (Schuetzle et al. 1981, 1982), woodstove samples
(Alfhelm et al. 1984) and ambient air particles (Eisenberg 1978; Ramdahl et
al. 1982).
HPLC offers several advantages, including higher resolution and lower
operating temperature than gas chromatography. The method is nondestructive,
and the injected sample can be recovered easily. This technique is gaining
wide acceptance as a modern method for the separation of organics in POM,
although the separation of isomeric PAHs from real-life samples may not be
complete. Further research is needed in this area. Caution must be exercised
before emphasizing the HPLC method for the fractionation of complex environ-
mental samples in the place of other conventional fractionation techniques
discussed earlier.
25
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c
0rs
26
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REFERENCES
Adams J, Menzies K, Levins P. 1977. Selection and evaluation of sorbent
resins for the collection of organic compounds. EPA-600/7-77-044, Office of
Research and Development, Washington, DC.
\
Ahlberg M, Berghem L, Nordberg G, Persson SA, Rudling L, Steen B. 1983.
Chemical and biological characterization of emissions from coal- and oil-
fired power plants. Environ. Health Perspect. 47:85-102.
Alfheim I, Holler M. 1979. Mutagenicity of long-range transported
abmospheric aerosols. Sci. Total Environ. 13:275-278.
Alfheim I, Lofroth G, Moller M. 1983a. Extracts of embient particulate
matter. Environ. Health Perspect. 47:227-238.
Alfheim I, Bergstrom JGT, Jenssen D, Moller M. 1983b. Mutagenicity in
emissions from coal- and oil-fired boilers. Environ. Health Perspect. 47:
177-187.
Alfheim I, Becher G, Hongslo JK, Ramdahl T, 1984. Mutagenicity testing of
high performance liquid chromatography fractions from wood stove emission
samples using a modified Salmonella assay requiring smaller sample volumes.
Environ. Mutagenesis 6:91-102.
Alfheim I, Ramdahl T. 1984. Contribution of wood combustion to indoor air
pollution as measured by mutagenicity in Salmonella and polycyclic aromatic
hydrocarbon concentrations. Environ. Mutagenesis 6:121-130.
Alfheim I, Lindskog A. 1984. A comparison between different high volume
sampling systems for collecting ambient airborne particles for mutagenicity
testing and for analysis of organic compounds. Sci. Total Environ. 34:203-
222.
Ames BN. 1971. The detection of chemical mutagens with enteric bacteria.
In: Hollaender A, ed. Chemical mutagens, principles and methods for their
detection, Vol. 1, New York: Plenum Press, pp. 267-282.
Ames BN, McCann J, Yamasaki E. 1975. Methods for detecting carcinogens and
mutagens with the Salmonella mammalian-microsome mutagenicity test. Mutat.
Res. 31:347-363.
Bartle KD, Lee ML, Novotny M. 1974. High-resolution GLC profiles of urban
air pollutant polynuclear aromatic hydrocarbons. Int. J. Environ. Anal. Chem.
3:349-356.
27
-------
Bartle KD, Lee ML, Novotny M. 1976. An integrated approach to the analysis of
Analyt Div\ ctem! Soc
Bjorseth A 1978. Analysis of polycyclic aromatic hydrocarbons in environ-
mental samples by glass capillary gas chromatography. In: Carcinogenesis?
3^ jones pw
Bogema M Monkmeyer PL. I960. The quadrant edge orifice-a fluid meter for
low Reynolds numbers. J. Basic Eng. 82:729.
v °f Polyamide for the separation of different polycyclic
387-389 hydr°Carbons ^ th*n-layer chromatography. J. Chromatogr. 130:
Brocco D, Cimmino A, Possanzini M. 1973. Determination of aza-heterocyclic
of thin-iayer and
Broddin G, Cautreels W, van Cauwenberghe K. 1980. On the aliphatic and
*nHy™°T Jh hydr°carbon levels ln "rtan and background aerosols from Belgium
and The Netherlands, Atmosph. Environ. 14:895-910.
Brusick DJ, Young RR. 1981. IERL-RTP Procedures Manual: level 1 environ-
mental assessment biological tests. EPA-600/ 8-81-024, U. S. Environmental
Protection Agency, Office of Research and Development, Washington, D.C.
Brusick DJ, Young RR. 1982. IERL-RTP manual: level 1 environmental assess-
ment, biological tests. EPA-600/S8-8 1-024, U. S. Environmental Protection
Agency, Industrial Environmental Research Laboratory, Research Triangle Park,
WLi •
Burchfield HP, Green EE, Wheeler RJ, Bllledeau SM. 1974. Recent advances in
the gas and liquid chromatography of fluorescent compounds. I. A direct gas-
phase isolation and injection system for the analysis of polynuclear arenes in
air particulates by gas-liquid chromatography. J. Chromatogr. 99:697-708.
Bursey JT, Smith J, Bunch R, Williams R, Berkley R, Pellizzari E. 1977
Application of capillary GC /MS /computer techniques to identification and
°f °rganlc comP°nents in environmental samples. Am. Lab. (Sept.
Calvert JG, Pitts JN. 1966. Photochemistry. New York: John Wiley and Sons,
Inc., pp. 366-557.
Candeli A, Morozzi G, Paolacci A, Zoccolillo L. 1975. Analysis using thin-
layer and gas liquid chromatography of polycyclic aromatic hydrocarbons in the
exhaust product from a European car running on fuels containing a range of
concentrations of these hydrocarbons. Atmos. Environ. 9:843-849
28
-------
Cantuti V, Gartoni GP, Libert! A, Torri AG. 1965. Improved evaluation of
polynuclear hydrocarbons in atmospheric dust by gas chromatography. J.
Chromatogr. 17:60-65.
Cautreels W, Van Cauwenberghe K. 1976. Extract ion of organic "^P°^ds from
airborne particulate matter: Water, Air, and Soil Pollution 6:103-110.
Chakraborty BB, Long R. 1967. Gas chromatography analysis of P°lycyclic
aromatic hydrocarbons in soot samples. Environ. Sci. Technol. 1:828-834.
Chatot G, Dangy-Caye R, Fontanges R. 1972a. Determination of the correction
factor in the estimation of polynuclear aromatic hydrocarbons separated by
thin-layer chromatography. Chromatographia 5:460-463.
Chatot G, Dangy-Caye R, Fontanges R. 1972b. Atmospheric P0^0"? b£*°
carbon. II. Application of chromatographic coupling in gas phase thin-layer
chromatography to the determination of polynuclear arenes. J. Chromatogr. 72.
202-206.
Chrisp CE, Fisher GL. 1980. Mutagenicity of airborne particles. Mutat. Res.
76:143-164.
Cleland JG 1981. Environmental hazard rankings of pollutants generated in
coal gasification processes. EPA-600/S7-81-101, U. S. Environmental
Protection Agency, Industrial Environmental Research Laboratory, Research
Triangle Park, NC.
Colucci JM. Begeman CR. 1971. Polynuclear Aromatic Hydrocarbons and other
pollutants in Los Angeles air. Proc. Int. Clean Air Congr. 2nd, Englund HM
(ed.), New York: Academic Press, pp. 28-35.
Commoner B, Madyastha P, Bronsdon A, Vithayathil AJ 1978. Environmental
mutagens in urban air particulates. J. Toxicol. Environ. Health 4:59-77.
Cuoitt IT 1980. Fate of toxic and hazardous materials in the air environ-
ment. EPA-600/S3-80-084, U. S. Environmental Protection Agency, Environmental
Sciences Research Laboratory, Research Triangle Park, NC.
Daisey JM, Hawryluk I, Kneip TU, Mukai F. 1979. Mutagenic activity in organic
fractions of airborne particulate matter. In: Novakov T. (ed.) Conference
on Carbonaceous Particles in the Abmosphere. Natl. Techn. Inform.
Serv., U. S. Department of Commerce, pp. 187-192.
Daisey JM, Allan CF, McGarrity G, Alterholt T, Louis J, McGeorge L, Lioy PJ.
1983. Effects of filter type on the organic composition and mutagenicity ot
inhalable particulate matter. Aerosol Sci. Technol. 2:295.
Dehnen W, Pitz N, Tomingas R. 1977. The mutagenicity of airborne particulate
pollutants. Cancer Lett. 4:5-12.
29
-------
« f- ',, TTS ' PltZ N' 1981' Studies on the -wtagenic effect of
extracts derived from airborne particulates within one Year on differently
351-366? "^ USing ^ AmeS teSt> ZM< Bakt' Hy8' l' Abt" °rig' B" l12''
C*romat°8raPhic analysis of polynuclear aromatic
°1UmnS: AppliCati°" t0 ^Pen-ion studies.
Dong M Locke DC, Ferrand E. 1976. High pressure liquid chromatographic
in ^ f°J H I?* T ySlS °f maj°r Parent P^ylic aromatic hydrocarbons
in suspended particulate matter. Anal. Chem. 48:368-372.
Dong M, Locke DC, Hoffmann D. 1977a. Characterization of aza-arenes in
11: °612-618C P°rtl0n °f susPetlded Particulate matter. Environ. Sci. Technol.
Dong M. Locke DC, Hoffmann D. 1977b. Separation of aza-arene by high pressure
liquid chromatography. J. Chromatogr. Sci. 15:32-35.
Drummond JG, Fenters JD, Ehrlich R, Aranyl C, Schiff L, 1982. Short-term
screening test for quick responses to pollutants where health effects data are
lacking EPA-800/S1-82-004, U. S. Environmental Protection Agency, Health
Effects Research Laboratory, Research Triangle Park, NC.
Duce RA. 1978. Speculations on the budget of particulate and vaporphase
116~244-273 °r8aniC Carb°n *" ^ gl°bal tr°P°sPhere« Pure APPl- Geophys.
Eisenberg WC. 1978. Fractionation of organic material extracted from
suspended air particulate matter using high pressure liquid chromatographv.
J. Chromatogr. Science 16:145-151. "
Epler JL. 1980. The use of short-term tests In the isolation and
identification of chemical mutagens in complex mixtures. In: deSerres JT,
Hollaender A. (eds.), Chemical Mutagens: Principles and Methods for their
Detection. New York: Plenum Press, Vol. 6, pp. 239-270.
Epler JL, Clark BR, Ho C-H, Guerin MB, Rao TK. 1978. Short-term bioassay of
complex organic mixtures: Part II, mutagenicity testing. In: Water ME et
al. (eds.), Application of short-term bioassays in the f ractionation and
269-289S °f C°mpleX environmental mixtures. New York: Plenum Press, pp.
Eppig CP, de Fillippi RP, Murphy RA. 1982. Supercritical fluid regeneration
Annaco of ^Carb°n US6d f°r volatile-°rganic-compound vapor adsorption. EPA
6UO/S 2-82-067, U. S. Environmental Protection Agency, Industrial Environmental
Research Laboratory, Research Triangle Park, NC.
Federal Register. 1971. No. 84.36, pp. 8191-8194.
30
-------
Fennelly PF. 1975. Primary and secondary participates as pollutants. J. Air
Pollut. C. Assoc. 25(7):697-704.
Fishbein L. 1976. Abmospheric mutagens. In: Hollaender A, (ed.), Chemical
mutagens, principles and methods for their detection, Vol. 4, New York.
Plenum Press, pp. 219-319.
Fitz DR, Doyle GJ, Pitts JN Jr. 1983. An ultrahigh volume sampler for the
multiple filter collection of respirable particulate matter. J. Air Poll.
Cont. Assoc. 33:877-879.
Fitz DR, Lokensgard DM, Doyle GJ. 1984. Investigation of filtration
artifacts when sampling ambient particulate matter for mutagen assay.
Atmospheric Environment. 18:205-213.
Fitzpatrick MB, Warner PO, Thiel DP, Lubs PL, Kerfoot EJ. 1983. Sampling and
analytical determination of airborne N.N-dimethylcyclohexyl-amine. Am. Ind.
Hyg. Assoc. J. 44:425-427.
Flessel P, Guirguis G, Cheng J, Chang K, Hahn E, Chan R, Ondo J, Fenske R,
Twiss S, Vance W, Wesolowski J. 1984. Monitoring of mutagens and carcinogens
in community air. Final report on the contract No. ARE Al-029-32, Research
Division, California Air Resources Board, Sacramento, CA.
Flessel P, Wesolowski JJ, Twiss S, Cheng J, Ondo J, Monto N, Chan R. 1981.
Integration of the Ames bioassay and chemical analysis in an epidemiological
cancL incidence study. In: Waters MD et al. (eds.), Short-term bioassays in
the analysis of complex environmental mixtures II, New York: Plenum
Publishing Corporation.
Fukino H, Mumura S, Inoue K, Yamane Y. 1982. Mutagenicity ot air particles.
Mutat. Res. 102:237-247.
Gehrs CW, Parkhurst BR, Shriner DS. 1978. Environmental testing. In:
Waters, MD et al. (eds.) Application of short-term bioassays in the
fractionation and analysis of complex environmental mixtures. New York:
Plenum Press, pp. 317330.
Gibson TL. 1982. Nitro derivatives of polynuclear aromatic hydrocarbons in
airborne and source particulate matter. Atmos. Environ. 16:2073-2076.
Gibson TL. 1983. Sources of direct-acting nitroarene mutagens in airborne
particulate matter. Mutat. Res. 122:115-121.
Gould RF. 1976. Photochemical smog and ozone reactions. In: Advances in
Chemistry Series 1134, Washington DC, American Chemical Society, p. 285.
Griest WH, Kubota H, Guerin MB. 1975. Resolution of polynuclear aromatic
hydrocarbons by packed column GLC. Anal. Lett. 8(12):949-957.
31
-------
1°' S"?^ndt A' B°hnke H> 19?2' SamPlln8 ^d analysis of poly-
cyclic aromatic hydrocarbons in automobile exhaust gas. Erdoel und Kohle,
Erdgas, Petrochemie vereinigt mit Brennstoff-Chemie 25:442-447.
Grimsrud EP Rasmussen RA. 1975. The analysis of chlorofluorocarbons in
9:1010-1013 83S chromat°8raPhy-mass spectrometry. Atmos. Environ.
Grob K, Grob GJ. 1971. Gas-liquid chromatographic-mass spectrometric
investigation of C6-C20 organic compounds in an urban atmosphere. J.
Chromatogr. 62:1-13.
Guerin MB, Clark BR, Ho C-H, Epler JL, Rao TK. 1978. Short-term bioassay of
complex organic mixtures: Part I, Chemistry. In: Waters MD et al. (eds.),
Application of short-term bioassays in the fractionation and analysis of
complex environmental mixtures. New York: Plenum Press, pp. 247-268.
Guidelines: Air Quality Surveillance Networks. 1971. U.S. EPA, Office of
Air Programs, Research Triangle, NC., Publication No. AP-98 (May).
Harke HP, Schuller D, Klimisch HI, Meisner K, 1976. Investigations of poly-
162*291-297 ° hydr°Carbons in Cl8arette smoke. Z. Lebensm. Unter.-Forsch.
Harnden DG. 1978. Relevance of short-term carcinogenicity tests to the studv
of the carcinogenic potential of urban air. Environ. Health Perspect.
-------
Hermann TS. 1974. Development of sampling procedures for polycyclic organic
matters and polychlorinated biphenyls. NTIS No. PB243362 (August).
Hill HH Jr., Chan KW, Karasek FW. 1977. Extraction of organic compounds from
airborne particulate matter for gas chromatographic analysis. J. Chromatogr.
131:245-252.
Hirayama T, Nohara M, Ando T, Tanaka M, Kitniko, Fukui S. 1983. Mutagenicity
of coal-pyrolyzed products and their photochemical reaction products with
nitrogen oxides in Salmonella typhumurium TA98 and TA100. Mutat. Res.
122:273-278.
\
Kites RA. 1975. Presented at the Division of Petroleum Chemistry, Inc.,
American Chemical Society, Chicago, Illinois, pp. 824-828.
Hoffman P, Norpoth K, Wickramasinghe RH, Muller G. 1980. The detection of
mutagenic air pollutants from filter samples by the Salmonella/mammalian S9
mutagenicity test (Ames test) with S. typhimurium TA98 (Part 1). Zbl. Bakt.
Hyg. I. Abt. Orig. B 171:388-407.
Howie SJ, Koesters EW. 1983. Ambient acrylonitrile levels near major acrylo-
nitrile production and use facilities. EPA-600/S4-83-016, U. S. Environmental
Protection Agency, Environmental Monitoring Systems Laboratory, Research
Triangle Park, NC.
Hughes TU, Pellizzari E, Little L, Sparacino C, Kolber A. 1980. Ambient air
pollutants: collection, chemical characterization and mutagenicity testing.
Mutat. Res. 76:51-83.
Huisingh J, Bradow R, Jungers R, Claxton L, Zweidinger R, Tejada S, Bumgarner
J, Fuffield F, Waters M, Summon V, Hare C, Rodriguez C, Snow L. 1978.
Application of short-term bioassays to the characterization of diesel particle
emissions. In: Waters MD et al. (eds.), Application of short-term bioassays
in the fractionation and analysis of complex environmental mixtures. New
York: Plenum Press, pp. 381-418.
Hunter L. 1975. Quantitation of environmental hydrocarbons by thin-layer
chromatography: Gravimetry/densitometry comparison. Environ. Sci. Technol.
9:241-246.
Janini GM, Johnston K, Zielinski WL Jr. 1975. Use of a nematic liquid crystal
for gas-liquid chromatographic separation of polyaromatic hydrocarbons. Anal.
Chem. 47:670-674.
Janini GM, Muschik GM, Schroer JA, Zielinski WL Jr. 1976. Gas-liquid
chromatographic evaluation and gas-chromatography/mass spectrometric
application of new high temperature liquid crystal stationary phases for
polycyclic aromatic hydrocarbon separations. Anal. Chem. 48:1879-1883.
Jones PW, Giammar RD, Strup PE, Stanford TB. 1976. Efficient collection of
polycyclic organic compounds. Environ. Sci. Technol. 10(8):806-810.
33
-------
Katz M, Pierce RC. 1976. Quantitative distribution of polvnuclear aromatic
hydrocarbons in relation to particle size of urban particulates. In: R.I
Freudenthal and P.W. Jones (eds.), Carcinogenesis, Vol. 1. Polynuclear
aromatic hydrocarbons: Chemistry, Metabolism and Carcinigensis. New York-
Raven Press, pp. 413-429.
Kebbekus BE, Bozelli JW. 1983. Volatile organic compounds in the ambient
atmosphere of the New Jersey, New York area. EPA-600/S3-83-022, U S
Environmental Protection Agency, Environmental Sciences Research Laboratory,
Research Triangle Park, NC. • r»
?J8e?oR*I ™9?4' GaS chromat°8raPhy ^ environmental,analysis. J. Chromatog.
OCX • I/!3D"~3;7.
Kohan M, Claxton L. 1983. Mutagenicity of diesel-exhaust particle extract,
1-nitropyrene, and 2,7-dinitrofluorenone in Salmonella typhimurium under
various metabolic activation conditions. Mutat. Res. 124:191-200.
Kolber AR, Hughes TU, Wolff TU, Little UW, Sparacino CM, Pellizzari ED.
1983. Development and assessment of procedures for collection, chemical
characterization and mutagenicity testing of ambient air. EPA-600/S2-83-045,
U. S. Environmental Protection Agency, Health Effects Research Laboratory,
Research Triangle Park, NC.
Krishna G, Ong T, Whong W-Z, Nath J. 1983a. Mutagenicity studies of ambient
airborne particles. I. Comparison of solvent systems. Mutat. Res. 124:
j. JL J"~ 1. ^ U •
Krishna G, Nath J, Whong W-Z, Ong T. 1983b. Mutagenicity studies of ambient
airborne particles. II. Comparison of extraction methods. Mutat. Res. 124:
12.1 — 128 •
Kubitschek HE, Venta L. 1979. Mutagenicity of coal fly ash from electric
power plant precipitators. Environ. Mutagenesis 1:79-82.
Kubitschek HE, Williams DM. 1980. Mutagenicity of fly ash from a fluidized-
bed combuster during start-up and steady operating conditions. Mutat. Res.
//•*.o/""^91»
Lane DA, Moe HK, Katz M. 1973. Analysis of polynuclear aromatic hydrocarbons,
some heterocyclics, and aliphatics with a single gas chromatograph column.
Anal. Chem. 45:1776-1778.
Lane DA, Katz M. 1977. Fate of pollutants in the air and water environments.
Part 2, Suffet IH (ed.), New York: Wiley-Interscience, pp. 137-154.
Lao RC, Thomas RS, Monkman JL. 1975. Computerized gas chromatographic-mass
spectrometric analysis of polycyclic aromatic hydrocarbons in environmental
samples. J. Chromatogr. 112:681-700.
Leighton PA. 1961. Photochemistry of air pollution. New York: Academic
Press.
34
-------
Levins PL. 1982. Environmental assessment measurement methods for organic
species. EPA-600/S7-82-040, U. S. Environmental Protection Agency, Industrial
Environmental Research Laboratory, Research Triangle Park, NC.
Lewtas J. 1983. Evaluation of the mutagenicity and carcinogenicity of motor
vehicles emissions in short-term bioassays. Environ. Health Perspect. 47:
141-152.
Liberti A, Morozzi G, Zoccolillo. 1975. Comparative determination of
polynuclear hydrocarbons in atmospheric dust by gas liquid chromatography
and spectrophotometry. Ann. Chim. 65:573-580.
\
Lindskog A. 1983. Transformation of polycyclic aromatic hydrocarbons during
sampling. Environ. Health Perspect. 47:81-84.
Lockard JM, Viau CJ, Lee-Stephens C, Cladwell JC, Wojciechowski JP, Enoch HG,
Sabharwal PS. 1981. Induction of sister chromatid exchanger and bacterial
revertants by organic extracts of airborne particles. Environ. Mutagen. 3:
671-681.
Lofroth G. 1978. Mutagenicity assay of combustion emissions. Chemosphere
10:791-798.
Loprieno N, Barole R, Baroncelli S, Bauer C, Bronzetti G, Cemmellini A,
Cercignani G, Corsi C, Gervasi G, Leporini C, Nierri R, Rossi A, Stretti G,
Turchi G. 1976. Evaluation of the genetic effects induced by vinyl chloride
monomer (VCM) under mammalian metabolic activation: studies in vitro and in
vivo. Mutat. Res. 40:85-96.
Lunde G, Bjorseth A. 1977. Polycyclic aromatic hydrocarbons in long-range
transported cerosols. Nature 268:518-519.
Lutz GA. 1982. Literature review of personal air monitors for potential use
in ambient air monitoring of organic compounds. EPA-600/S4-82-048, U. S.
Environmental Protection Agency, Environmental Monitoring Systems Laboratory,
Research Triangle Park, NC.
Ma T, Isbandi D, Khan S, Tseng Y. 1973. Low level of S02 enhanced chromatid
aberrations in Tradescantia pollen tubes and seasonal variation of the
aberration rates. Mutat. Res. 2:93-100.
Malaveille C, Bartsch H, Barbin A, Camus M, Montesano R. 1975. Mutagenicity
of vinyl chloride, chloroethyleneoxide, chloroacetaldehyde and chloroethanol.
Biochem. Biophys. Res. Commun. 63:363-370.
Malaveille C, Planceh G, Bartsch H. 1976. Factors for .efficiency of the
Salmonella/microsome mutagenicity assay. Chem.-Biol. Interact. 12:251-263.
Mitera J, Novak J, Mostecky J. 1977. The determination of organic substances
in the atmosphere. Sbornik Vysoke Skoly Chemicko Technologicke V Prage,
Technologic Paliv 36:5-48.
35
-------
of trace
of air samples
Hakagawa B, Kltamori S, Horlkawa K, Nakashlma K, Toklwa H, 1983. Identifica-
tion of dlnitropyrenes In dlesel-exhaust particles. Their probable presence as
the major mutagens. Mutat. Res. 124:201-211. presence as
\
(NAS)' 1972' Particulate polycyclic coganic
National Research Council (NRC). 1976. Vapor-phase organic pollutants
National Academy of Sciences, Washington, DC, pp. 1-411?
p « . Baltimore:
Park Press, pp. 1-343.
Novotny M, Lee ML Barf-IP K"n 107/1 TU« -, ..u j c *
j , s. ii.u, UC1J.I.J.C ro^. iy/n, me methods for fractionation analvtical
mJxa^a^°n f d *dentiflcat*on °f Polynuclear aromatic hydrocarbons in complex
mixtures. J. Chromatogr. Sci. 12:606-612.
Ohnishi Y, Kachi K, Sato K, Tahara I, Takeyoshi H, Tokiwa H. 1980. Detection
ol mutagenic activity in automobile exhuast. Mutat Res. 77:229-240.
OSHA. 1976 OSHA Safety and Health Standards, General Industry. Occupational
Safety and Health Administration, 29CFR1910, OSHA 2206. ccupacionai
Panalaks T. 1976. Determination and identification of polycyclic aromatic
hydrocarbons in smoked and charcoal-broiled food products by high pressure
liquid chromatography. J. Environ. Sci. Hlth. Bll(4):299-315.
Pellizzari ED. 1974. Development of method for carcinogenic vapor analysis
in ambient atmospheres. EPA-650/2-74-121, U. S. Environmental Protection
Agency, Office of Research and Development, Washington, DC.
Pellizzari ED. 1975. Development of analytical techniques for measuring
ambient atmospheric carcinogenic vapors. EPA-600/2-75-076, U. S. Environmental
Protection Agency, Office of Research and Development, Washington, DC.
Pellizzari ED. 1977a. Analysis of organic air pollutants by gas chromato-
graphy and mass spectrometry. EPA- 600/2-77-100, U. S. Environmental
Protection Agency, Office of Research and Development, Washington, DC.
Pellizzari ED. 1977b. The measurement of carcinogenic vapors in ambient
atmospheres. EPA-600/7-77-055, U. S. Environmental Protection Agency, Office
of Research and Development, Washington, DC.
36
-------
Pellizzari ED. 1978. State-of-the-art analytical techniques for ambient vapor
phase organics and volatile organics in aqueous samples from energy-related
activities. In: Waters MD (eds.) Application of short-term bioassays in the
fractionation and analysis of complex environmental mixtures. New York:
Plenum Press, pp. 195-225.
Pellizzari ED, Little LW, Sparacino C, Hughes YI, Claxton L, Waters MD. 1978.
Integrating microbiological and chemical testing into the screening of air
samples for potential mutagenicity. In: Waters MD, et al. (eds.).
Application of short-term bioassays in the fractionation and analysis of
complex environmental mixtures. New York: Plenum Press, pp. 333-351.
Pellizzari E, Demian B, Schindler A, Lam K, Jeans W, 1983. Preparation and
evaluation of new sorbents for environmental monitoring, Volume I and Volume
II. EPA-600/S4-83-015 a fie b, U. S. Environmental Protection Agency,
Environmental Monitoring Systems Laboratory, Research Triangle Park, NO.
Pitts JN, Jr. 1983. Formation and fate of gaseous and particulate mutagens
and carcinogens in real and simulated abmospheres. Environ. Health Perspect.
47:115-140.
Pitts JN, Grosjean D, Mischke TM. 1977. Mutagenic activity of airborne
particulate organic pollutants. Toxicol. Lett. 1:65-70.
Preidecker BL. 1980. Comparative extraction of Houston air particulates^with
cyclohexane or a mixtue of benzene, methanol and dichloromethane II. Environ.
Mutagen. 2:85-88.
Ramamoorthy MV, Seetharamiah K. 1966. Quadrant-edge orifice nd performance
at very high Reynolds numbers. J. Basic Eng. 88:9.
Ramdahl T, Becher G, Bjorseth A. 1982. Nitrated polycyclic aromatic hydro-
carbons in urban air particles. Environ. Sci. Technol. 16:861-865.
Rannug V. 1983. Data from short-term tests on motor vehicle exhausts.
Environ. Health Perspect. 47:161-169.
Rannug W, Gothe R, Wachtmeister C. 1976. The mutagenicity of chloroethylene
oxide, chloroacetaldehyde, 2-chloroethanol and chloroacetic acid, conceivable
metabolites of vinyl chloride. Chem. Biol. Interact. 12:251-263.
Rappaport SM, Wang YY, Wei ET. 1980. Isolation and identification of a
direct-acting mutagen in diesel-exhaust particulates. Environ. Sci. Technol.
14:1505-1509.
Rosenkrantz H, McCoy EC, Anders M, Speck WT, Bickers D.. 1978. The use of
microbial assay systems in the detection of environmental mutagens in complex
mixtures. In: Waters MD et al., eds. Application of short-term bioassays in
the fractionation and analysis of complex environmental mixtures. New York:
Plenum Press, pp. 333-351.
37
-------
Sampling Location Guidelines. 1972. U. S. Environmental Protection Agency
Div. of Abmospheric Surveillance (November). Agency,
Sawicki E, Meeker JL, Morgan MU. 1965. The quantitative composition of air
pollution source effluents in terms of aza-heterocyclic compounds and
polynuclear aromatic hydrocarbons. Int. J. Air and Water Pollut! 9:291-298.
Sawicki E, Stanley TW, McPherson S, Morgan M. 1966. Use of gas-liquid and
yrah ir Pollutants by8f luoro'metry
separation of^l*' T* ™' ^ ' ***** and thi^^r electrophoretic
3^:522-527. P°lynuclear aza-beterocyclic compounds. J. Chromatogr.
Sawicki E 1970. Analysis of aerotoxicants. CRC Critical Reviews in
Analytical Chemistry (November).
' G1SClard JB> Monkma* JL. Nelian RE, Ripperton
L1970a T . > . , pperto
LA. 1970a. Tentative method of microanalysis for benzo(a)pyrene in airborne
particulates and source effluents. Hlth. Lab. Sci. 7:56-59 airborne
T,n,r f '/1SClard JB« Monkman JL. Neligan RE, Ripperton
Tentative method of spectrophotometric analysis for
benZo(a)pyrene in atmospheric particulate matter. Hlth. Lab. Sci. 7
(suppi; : 68-71.
E' Glsclard JB> Monkman JL, Neligan RE, Ripperton
ct f method Of a^y^ f°r polynuclear aromatic nydrocarbon
content of atmospheric particulate matter. Hlth. Lab. Sci. 7(suppl): 31-44.
'TO' e f ye DL' M°nkman •"" ^smussen RA, Ripperton
White LO. 1975. Total particulate aromatic hydrocarbons in air:
Ultrasonic extraction method. Hlth. Lab. Sci. 12:407-414.
Sawicki E. 1978. Abmospheric genotoxicants — what numbers do we collect?
In: Waters MD et al., eds. Application of short-term bioassays in the
fractionation and analysis of complex encironmental mixtures. New York-
Plenum Press, pp. 171-194 i«"..
Schuetzle D, Lee FSC, Prater TU, Tejeda SB. 1981. The identification of
polynuclear aromatic hydrocarbons (PAH) derivatives in mutagenic fractions of
diesel particulate extracts. Int. J. Environ. Anal. Chem. 9:93-144.
Schuetzle D Riley TL, Prater TI, Harvey M, Hunt DF. 1982. Analysis of
54:265^27?? ar°m^ic hydrocarbons in diesel particulates. Anal. Chem.
Schuetzle D. 1983. Sampling of vehicle emissions for chemical analysis and
biologicla testing. Environ. Health Perspect. 47:65-80.
38
-------
Schultz MI, Orheim RM, Bovee HH. 1973. Simplified method for the
determination of benZo(a)pyrene in ambient air. Amer. Ind. Hyg. Assoc. J.
34(9):404-408.
Sexton N, Myers L, Hughes T. 1981. A plan to develop and implement a quality
assurance program forthe Ames/ Salmonella test. EPA-600/S2-81-054, U. S
Environmental Protection Agency, Health Effects Research Laboratory, Research
Triangle Park, NC.
Shushunouo AF, Shkodich PE, Shkanakin NG, Lembik LZH. 1975. Gas chromagraphic
determination of polycyclic aromatic hydrocarbons in sewage. Gig. banit.
8:61-62.
Singh HB, Salas LJ, Smith A, Stiles R, Shigeishi H 1981. Atmospheric
measurements of selected hazardous organic chemicals. EPA-600/S3-81-032,
U. S. Environmental Protection Agency, Environmental Sciences Research
Laboratory, Research Triangle Park, NC.
Singh HB, Salas LJ, Stiles R, Shigeishi H. 1983. Measurements of hazardous
organic chemicals in the ambient atmosphere. EPA-600/S3-83-002, U. S.
Environmental Protection Agency, Environmental Sciences Research Laboratory,
Research Triangle Park, NC.
Stenberg U, Alsberg T, Westerholm R. 1983a. Applicability of a cryogradient
technique for the enrichment of PAH from automobile exhausts: demonstration
of methodology and evaluation experiments. Environ. Health Perspect.
47:43-51.
Stenberg U, Alsberg T, Westerholm R. 1983b. Emission of carcinogenic
components with automobile exhausts. Environ. Health Perspect. 47:53-63.
Strup PE, Giammar RD, Stanford TB, Jones PW. 1976. In: Carcinogenesis, Vol.
1, Polynuclear aromatic hydrocarbons: chemistry, metabolism and carcino-
genesis, Freudenthal RI, Jones PW (eds.), Raven Press, New York, pp. 241-251.
Talcott R, Wei E. 1977. Airborne mutagens bioassayed in Salmonella
tyrhimurium. J. Natl. Cancer Inst. 58:449-451.
Talcott R, Harger W, 1979. Mutagenic activity of aerosol size fractions. EPA
Publication 600/3-79-032, U. S. Environmental Protection Agency, Office of
Research and Development, Washington, DC.
Teranishi K, Hamada K, Watanabe H. 1978. Mutagenicity in Salmonella
typhimurium mutants of the benzene-soluble organic matter derived from
airborne particulate matter and its five fractions. Mutat. Res. 56:273-280.
Thilly WG, Longwell J, Andon BM. 1983. General approach to the biological
analysis of complex mixtures. Environ. Health Perspect. 48:129-136.
Tokiwa H, Morita K, Fakeyoshi A, Tokahashi K, Ohnishi Y. 1977. Detection of
mutagenic activity in particulate air pollutants. Mutat. Res. 48:237-248.
39
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ssaTof'extractrnf' ^^ K> .°h*lshl Y« ^80. Mutagenic and chemical
assay of extracts of airborne particulates. Mutat. Res. 77:99-108.
R\V°lt!ier G. Bednarik R. 1977. Direct fluorometric analysis of
The Li. of the
Tyson BJ. 1975. Chlorinated hybrocarbons in the atmosphere - analysis at the
parts per trillion level by GC/MS. Anal. Lett. 8:807-813. anaiysis at tne
G?d*l±™S: air «uality surveillance networks. Publication
^ *' *' ^"""^ *«»t.ctlon Agency,
U.S. EPA. 1972. Sampling location guidelines. Division of Atmospheric
Surveillance, U.S. Environmental Protection Agency, Research Triangle Park,
1 N vj •
Van Vaeck L, Broddin G, Cauwenberghes KV. 1980. On the relevance of air
pollution measurements of aliphatic and polyaromatic hydrocarbons in ambient
air particulate matter. Biomed. Mass Spectrometry 7:473-483.
Vouk VB, Piver WT. 1983. Metallic elements in fossil fuel combustion
products: amounts and form of emissions and evaluation of carcinogenicity and
mutagemcity. Environ. Health Perspect. 47:201-225.
Walling JF, Berkley RE, Swanson DH, Toth El. 1982. Sampling air for gaseous
ocn he"licals usin8 solld adsorbents. Application to Tenax. EPA-600/
co ocn . . -
S4-82-059, U. S. Environmental Protection Agency, Environmental Monitoring
Systems Laboratory, Research Triangle Park, NC.
Wang, CY, Lee MS, King CM, Warner PO. 1980. Evidence for nitroaromatics as
direct-acting mutagens of airborne particulates. Chemosphere 9:83-87.
Wang YY, Rappaport SM, Sawyer R, Talcott R, Wei ET. 1978. Direct-actine
mutagens in automobile exhaust. Cancer Lett. 5:39-47.
Ward DE, McMahon CK, Johansen RW. 1976. An update on particulate emissions
from forest fires. In: Emissions from forest burning-session 2. Proceedings
of the 69th Annual Meeting Air Pollution Control Association. Pittsburgh:
Air Pollution Control Association.
Waters MD, Nesnow S, Huisingh JL, Sandhu SS, Claxton L, eds. 1978. Application
of short-term bioassays in the fractionation and analysis of complex environ-
mental mixtures. New York: Plenum Press.
Wei ET, Shu HP. 1983. Nitroaromatic oarcinogens in diesel soot: a review of
laboratory findings. Am. J. Pub. Health 73:1085-1088.
Wei ET, Wang YY, Rappaport SM. 1980. Diesel emissions and the Ames test: a
commentary. J. Air Pollution Control Assoc. 30:267-271.
40
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West DA, Hodgson FN, Brooks JJ, DeAngelis DG, Desai AG, McMillin CR. 1981.
Pobential abtnospheric carcinogens: phases 2/3. Analytical technique and
field evaluation. EPA-600/S2-81-106, U. S. Environmental Protection Agency,
Environmental Sciences Research Laboratory, Research Triangle Park, NC.
White L, Taylor D, Mauer P, Kupel.. 1970. A convenient method for the
analysis of selected solvent vapors in the industrial atmosphere. Am. Ind.
Hyg. Assoc. 31:225-232.
Whong W-Z, Stewart J, Ong T. 1984. Development of an in situ microbial
mutagenicity test system for airborne workplace mutagens: laboratory evalua-
tion. Mutat. Res. 130:45-51.
Woidich H, Pfannhauser W, Blaicher G, Tiefenbacher K. 1977. Thin-layer
chromatographic separation and in situ detection of polycyclic ^omatic
hydrocarbon: Studies using acetylated cellulose. Chromatographia 10:140-146.
Zelac R, Cromroy H, Bolch W, Dunavant B, Bevis H. 1971. Inhaled ozone as a
mutgen. I. Chromosome aberrations induced in Chines Hamster lymphocytes.
Environ. Res. 4:262-282.
Zoccolillo L, Liberti A. 1976. Determination of polycyclic hydrocarbons by
channel thin-layer chromatography. J. Chromatogr. 120:485-488.
Zweidinger RB. 1982. Emission factors from diesel and gasoline-powered
vehicles: correlation with the Ames test. In: Lewtas J, (ed.) Toxico-
logical effects of emissions from diesel engines. Amsterdam: Elsevier,
pp. 83-96.
41
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AIR PARTICULATES WORKGROUP REPORT
Workgroup Tasks
Summary of Tasks —
Consider question of Vapor Phase and/or Particulate Organics.
technl«ues for ** particles. How much
How to prepare crude extracts for bioassay?
Key Peer Review Comments —
(addressed by protocol)
Summary of Workgroup Progress —
Mutagenicity protocols for VPOs - not available.
Air particle collection techniques are source dependent- thus a
genera collection technique cannot be specified in detail here However a
specific protocol for Hi-Vol sampling of ambient air particullte is described
A scheme for sample preparation protocol is enclosed (Figure 4)? deSCribed
Schemes for chemical f ractionation of crude air particulate extracts are
utM ^rVrth' specifvractirtion prot°c°i ca™ot i-^c«s
(at this time). Further research in LC/HPLC methodology is recommended.
Summary of Key Workgroup Discussions
Major Consensus Opinions —
Air protocol to focus on particles; VPOs will not be considered.
Air particle collection techniques are sufficiently source-dependent to
preclude development of a generalized particle collection protocol However
salSn^r r0" ^ 'f I6"' 3ir Partlcle ""^toring. the use of sta^ard H^Vol
sampling is recommended; i.e., community air particulate from 24-h Hi-Vol
sampler will provide sufficient sample for minimum characterization on
fnaly's ^ ^^ ^ teSt* bUt nOt e"°USh for detailed chemical
42
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Air Particulate
Sample on
High-Volume Filter
Sample Extraction
(Soxhlet Extraction
or Sonication)
Solvent Evaporation
(Using Rotary-Evaporator and/or
Kuderna-Danish Concentrator)
Residual
Mass
Determination
Chemical
Analyses
Solvent Exchange
with DMSO
Archived
Aliquots
Bioassay
Figure 4. Flow scheme for the preparation of samples
from ambient air particulates.
43
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•£==•= sx .
Unresolvable Issues —
Issue: Minimum Air Particulate Sample Size-»Sclentif ic» Basis
y' 12-nTso 7^^ S"ff*Cient P«tlc«late on standard Hi-Vol in 24-h
-"
Minimum Sample Size —
Workgroup Recommendation:
Use 24-h Hi-Vol for genotoxicity testing-routine monitoring.
Hi-Vol compositing required for full bioassay /chemical characterization
Use Ultra Hi-Vol sampler, but not feasible for routine applications.
Choice of Solvent —
Single solvent, sequential extraction, mixed solvent.
Recommend side-by-side evaluation.
Solvent Removal Steps —
introduLf hK~H 3n? S°11Vent exchan§e stePs' Are they necessary? Each
protocol? 8 Vel °f C°mPlexlt? - *°°* to obtain minimut/acceptable
steps!600™"611" C°mparlSOn ^th/without K-D and with/without solvent exchange
Fractionation Scheme —
Acid-Base-Neutral or LC-- methods to be developed for toxicity?
Emphasis of LC methodology— to be developed.
Evaluation of Proposed Protocol
Adequacy —
£„
44
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Validity—
To be validated.
Limitations—
Some steps in protocol require validation.
Comparison to Other Five Media Protocols—
Common problem of fractionation scheme.
Other Data or Information Requirements
Information Gaps— v
Sample collection: Hi-Vol "A," Ultra Hi-Vol "B."
Preparation of crude extracts. "B" - QC data needed.
1. Solvent extraction comparisons.
2. Solvent removal issues.
Fractionation of crude extracts. "C" - Research needed.
Research Program Needs—
Rationale for need - fractionation scheme necessary for toxic samples and
chemical characterization.
Alternatives and variables - not available.
45
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PROTOCOL FOR PREPARATION OF AIR PARTICULATES FOR MUTAGENICITY TESTING
1-0 Scope and Application
1.1 Introduction —
ir or source samples for Ames biotesting. Procedures for the
of
The procedures described are intended to be used under the direction
^z^r^: *
1.2 Scope and Application —
rseoranf the ^option/preparation of
applcations: "" PartlCulateS' Thls Protocol has the following
a. Routine air quality characterization to determine the
environmental quality (by EPA, Army, etc.).
b.
Routine monitoring of community air and observation of seasonal
trends in order to get data for correlating the geographic
distribution of cancer incidence with air pollutants.
!- ^he,methods P™P°sed in this protocol are limited to direct applica-
n * f* T "f1"" *±T Partlculate samples. Detailed procedures are
possiblf fo^ I" ^P iCaL10n> IndireCt aPPlicatio«. with modifications, is
1° r£* t0 m°S °ther amblent or source Particulate samples. Modifications
modJflr P™Cedure are not Deluded, since the types of samples and associated
modifications are source-specific and, while similar to the detailed pro-
of the air particuiates and the
2.0 Summary of Method
bVpii-h/^ paficles (1 8) are extracted twice with dichloromethane (100 mL)
by either Soxhlet extraction (5-10 cycles/hour for 16 hours at 40 C) or
sonication (at room temperature for 15 minutes each time) . The dichloro-
methane extract is filtered through a 0.5-ym Fluoropore filter (Mllipore
Corporation) into a round-bottom evaporating flask and concentrated tliO mL
46
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in either a rotary evaporator or a Kuderna-Danish concentrator. One portion
of this extract (5 mL) is saved for mass determination by gravimetric analysis
and chemical analyses. The other portion of the extract (5 mL) is solvent
exchanged with dimethyl sulfoxide (5 mL) under a gentle stream of nitrogen,
and the final volume is noted. This DMSO solution is further diluted to
appropriate concentration and bioassayed.
2.2 It is recommended that the air samples be stored in the dark at -20 C,
and the solvent (dichloromethane or DMSO) extracts in amber-colored Teflon
screw-capped bottles at -20 C. These air samples and solvent extracts should
be thawed to room temperature prior to use.
\
3.0 Definitions
3.1 Air Particulates—
Air particulates are defined operationally as air particles having
20-ym aerodynamic diameters.
Recent studies (Talcott and Harger 1979, Preidecker 1980) have shown
that most of the mutagenic activity associated with air particulate samples is
found in particles of £2-ym aerodynamic diameter. This is important when one
considers that the penetration of particles into the lung is inversely related
to particle size, i.e., most of the larger particles are removed in the upper
part of the respiratory tree (Hatch and Gross 1964). According to Pierce and
Katz (1975), approximately 70% to 90% of the total polynuclear aromatic
hydrocarbon (PAH) content of airborne particles is associated with particles
of <_5-ym diameter.
4.0 Interferences
4.1 Sampling Artifacts—
4.1.1 Non-incorporation of sampling efficiency— The evaluation of sampling
efficiency is particularly difficult under ambient atmospheric sampling
conditions. The percent recovery of a collected sample, however, is rarely
determined during either ambient or source sampling. The lack of incorpora-
tion of sampling efficiency lends considerable uncertainty in the reported POM
levels.
4.1.2 Evaporative losses and reactive conversion—Collection of POM on
widely used glass-fiber filter alone without the backup adsorbent is subject
to two kinds of errors. First, lower molecular weight compounds on adsorbed
particulate phase may have some equilibrium vapor concentrations under atmos-
pheric conditions. This vapor part will escape collection on the filter.
Second, air passing through a filter containing the collected substances will
carry away amounts equal to or less than the equilibrium vapor concentration
because of desorption from the adsorbed substrate. Pupp et al. (1974) deter-
mined the equilibrium vapor concentration for a number of polycyclic aromatic
hydrocarbons, and concluded that considerable losses occur during ambient
sampling of compounds having an equilibrium vapor concentration of 0.5 x
10 mg/m or higher at ambient temperature. Considerable losses are expected
with pyrene, anthracene, phenanthrene and benzo(a)anthracene.
47
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uor-
.
anthene. enz°apyrene is more prone to oxidation than benzo(b)fl
Pitts and co-workers (Pitts et al
!SSi^s^^~^
dioxide. ""^derivatives by exposure of aromatic compounds to nitrogen
4.2 Losses During Transportation and Storage—
4.3 Sample Preparation-
Several errors can be introduced during sample preparation stage.
pf-
48
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4.4 Other Sources of Error—
The use of all-glass (Pyrex) apparatus and glassware will minimize
error due to contaminations.
Appropriate solvent blanks have to be run simultaneously in order to
see whether any artifact has been introduced in the mutagenicity determination.
5.0 Safety
5.1 In vivo bioassays have demonstrated that the greatest carcinogenic
activity~of organic pollutants in air is associated with the fraction contain-
ing both polynuclear aromatic compounds and their nitrpgen analogs, the nitro-
and aza-arenes. Since the toxicity or carcinogenicity of chemicals used or
extracted in this method has not been precisely defined, each chemical or
extract should be treated as a potential health hazard, and exposure to these
chemicals or extracts should be minimized. Each laboratory is responsible for
maintaining awareness of OSHA (1976) regulations regarding the safe handling
of chemicals used in this method.
5.2 It is generally recommended that the laboratory personnel avoid
contact with air particulates and that all operation steps, such as Soxhlet
extraction or sonication, solvent evaporation, solvent exchange with dimethyl
sulfoxide (DMSO), etc., be carried out in a hood (A-type). Hot water baths
heated on electric heating mantles are recommended for solvent evaporation.
6.0 Apparatus and Equipment
A general list of the apparatus and equipment necessary for air sample
preparation is presented below. For many items, equivalent products are
available from other sources. Listing does not constitute a specific
endorsement.
6.1 Sampling—
6.1.1 High-Volume (Hi-Vol) particulate sampler (Sierra Instruments, Model
305-2000).
6.1.2 Glass-fiber filters (8 x 10 in or 20.3 x 25.4 cm EPA Grade, Vhatman).
6.1.3 Pallflex TX40H120-WW Teflon-coated filters.
6.2 Extraction and Sample Preparation—
6.2.1 Soxhlet extractor: 40-mm ID, with 500-mL round-bottom flask.
6.2.2 Sonicator (Bransonic, Models 220 or 32).
6.2.3 Kuderna-Danish (K-D) evaporative concentrator with a 3-ball Snyder
Column (Kontes, Vineland, New Jersey).
6.2.4 Rota-Vap evaporator (Kontes).
49
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S<:1-"»<= O-W. Scientific Product.. Inc.
Pro-
6.3 Miscellaneous —
-' 50°- - 1.0«HL (Fisher
6.3.4 Glass rods and stainless steel spatulas (Fisher Scientific Company).
7t° Reagents and Consumable Materials
7.1 Solvents —
e
redistilUnf eJf°-ai°r? In4tend8 *° use the purchased solvents without
TprVc. SP^ctral-frade or pesticide quality solvents (from: Burdick and
Jackson Laboratories or Fisher Scientific Company) include:
Dichloromethane (Methylene chloride)
Cyclohexane
Methanol
Dimethyl Sulfoxide (DMSO)
7.2 Disposable Items —
^ HI , Screw-caPPed Teflon vials (Fisher Scientific Company or Kontes)
Soxhlet extraction thimbles (Fisher Scientific Company o^Kontes) I 5-um
Fluoropore filters (Millipore Corporation) and latex surgical Jloves' (Pharma
Seal Laboratories) are included among the disposable items to be used
8>° Sample Collection. Preservation and Handling
8.1 Sample Collection —
sample size-specific. Elaborate description 'f the
50
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various source and ambient air sampling systems is beyond the scope of this
protocol. The reader is requested to refer to the relevant reference(s) in
this section.
8.1.2 Source sampling—Source sampling techniques include application of
impactors, cyclones, electrostatic precipitators and filter media to recover
particulate emissions with or without the ability to select the particle sizes
collected. Examples of source sampling systems include EPA Method 5, Source
Assessment Sampling Systems, Instack Cyclone Systems and Dilution Tunnel
Sampling. An overview of these sampling systems is briefly described in
IERL-RTP Procedures Manual: Level 1 Environmental Assessment Biological Test
(Brusick and Young 1981). A more detailed description is included in the
IERL-RTP Procedures Manual: Level 1 Environmental Assessment (Second Edition)
(Lentzen et al. 1978).
8.1.3 Ambient air sampling—Ambient air sampling techniques include the
standard high-volume (Hi-Vol) samplers (Federal Register 1971), massive-
volume samplers (Henry and Mitchell 1978), medium-volume samplers, low-volume
samplers and ultra high-volume samplers (Fitz et al. 1983).
The sampling of POM in ambient air is usually performed with the
objective of determining the particle size distribution and the nature and
concentrations of individual components at various points in the environment.
The selection of sampling sites plays an important role in sample collection.
Sampling sites are determined to evaluate the following: (1) characterization
of rural or urban background levels, (2) assessment of health hazards to
people in the vicinity, (3) determination of source effects and (4) establish-
ing of transport mechanisms. Reference should be made to the available
sources (USEPA 1971, 1972) for detailed treatment of these aspects in the
selection of sites.
In addition to the selection of a preferable method and site, certain
other factors must be adequately taken into consideration during ambient
sampling. The height of the sampler intake above ground level and the local
topography and climate influence the data obtained. For example, the in-
fluence of summer-like temperature on losses of benzo(a)pyrene from airborne
particles has been studied during real high-volume atmospheric samplings
(DeWiest and Rondia 1976). Seasonal variation in the specific surface areas
and densities of suspended particulate matter has been observed (Corn et al.
1971, Flessel et al. 1984). The effect of wind on the collection efficiencies
of particulate matter has also been demonstrated (Ogden and Wood 1975). Other
factors, such as sampling rate and time, are also important in the overall
sampling strategy (Flessel et al. 1984; Sweetman et al., 1984). The total
amount of samples to be collected usually depends on the amount necessary to
perform the bioassay. The optimum amount of air particulates needed for both
bioassay and chemical analyses is approximately 2 g. However, it is possible
to do the Ames bioassay with 100 mg of the starting material.
For routine monitoring of the total mutagenicity of POM, air samples
can be collected in standard Hi-Vol samplers on filters with inert filter
(Teflon) collection media (Flessel et al. 1981, 1984, Sweetman et al. 1984).
51
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Alfheim and Lindskog (1984) have recently compared different high-volume
sampling systems for collection of POM for muLgenicity testin?u.Ing Ames
In certain instances, it is desirable to collect larger amounts of
"ttel'of th "'T11 MOaSSay ^ detall6d Chemlcal -"lysis oJ the organic
in e'als ^ ^T^^^ **""' In addltl°n' Sh°rt tlme -flection
nr!!T I desirable to prevent evaporation or chemical reaction of
of the YrM°U? "' 8OTa °f WhlCh "^ be ™SP™S^* for the positive response
of the particulate extract in mutagen bioassay. The use of an ultrahigh-
volume sampler developed by Pit, et al. (1983) will solve these problems, and
it can also be used for monitoring the total mutagenicity of POM. However, it
6 «Pl« is^ot feasible for
*. A ?±U
-------
Figure 5. Assembled high-volume sampler and shelter
(Federal Register 1971).
53
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TO 5 cfm Pump
«tidB Presentation of connections for sorbent
cartridge for high-volume sampler (Lentzen et al. 1978).
54
-------
depends on the amount necessary to perform the bioassay. However, to obtain
the needed amount of organic fraction, it may become necessary to pool to-
gether organic fractions of several individual high-volume air samples from a
single monitoring site. Air should not be sampled for more than 24 hours in
order to avoid artifacts due to chemical reactions occurring on the filters.
The POM present in polluted air sorbed onto airborne particulate
matter is usually characterized as primary and secondary. Primary parti-
culate matter is found in sizes between 1 and 20 ym (Fennelly 1975).
Secondary particulate matter ranges in size from molecular clusters on the
order of 5 nm to particles with diameters as large as several micrometers
(Fennelly 1975). Very fine particles that cannot be retained on fiber-glass
filters act like a gas or vapor in that they follow fluid streamlines and are
subject to Brownian motion. Various absorbents have been used for collection
of this gaseous phase (Bertsch et al. 1974, Fox and Staley 1976, Krstulovic et
al. 1977, Schuetzle et al. 1973).
8.1.5 Distribution of particle size—An assessment of the POM content of the
polluted atmosphere with respect to the size of particles in an aerosol often
becomes necessary. Knowledge of the fraction of the air particulate matter
which can cause deposition in the various compartments of the respiratory
system is of prime importance since some of the POMs are proven carcinogens.
Size fractionation of particles is an important factor for the quantitation of
mutagenicity, since the smaller sized particles (<2 ym) present a greater
surface area for adsorption of the organic pollutants per unit mass of par-
ticulate. The smaller particles are more easily inhaled and deposited in the
lung and are more difficult to expel (Hughes et al. 1980, Kolber et al. 1983,
Lippmann et al. 1979, Natusch and Wallace 1974, Schlesinger and Lippmann 1978,
Yeh et al. 1976). An increase in mutagenicity with decreasing particulate
diameter (increased surface area) has been demonstrated for coal fly ash
(Fisher et al. 1979) and for air particulates (Commoner et al. 1978).
Whitby et al. (1974) have presented evidence that the mass distribu-
tion of atmospheric aerosols is usually bimodal, with one mode occurring below
1.0 ym and the other mode occurring in the 5- to 15-ym range. A dichotomous
sampler for particulates has been designed to collect and fractionate samples
into two size ranges (Dzubay and Stevens 1975). Membrane filters in the two
air paths collect the respective samples. However, such a sampling technique
has rarely been applied to collect POM from air.
The experimental determination of particulate distribution is fre-
quently done by five-stage Andersen Hi-Vol cascade impactors (Katz and Pierce
1976). The first four stages of the sampler constitute the fractionating
head, while the fifth stage is a backup filter positioned between the
fractionating head and a standard Hi-Vol air sampler. When operated at a flow
rate of 20 cfm, the sampler fractionates suspended particulate matter into
five aerodynamic size ranges according to certain cut-off diameters.
8.2 Preservation and Handling—
8.2.1 Air particulate samples—After sampling, filters are folded into
quarters (sample touching sample), held in glass-lined envelopes and trans-
55
-------
;:
-- -
^
Solvent extracts— All solvent (dichloromethane and DMSO) extracts
or SOT:? *? PrP5nrl? Ubeled Tefl0n — ^ amber-cofored battles
or 50-mL size) at -20 C. It is highly recommended that the DMSO extracts
to relTwith Li ^ ?** '^ ^ extracti- ^te, since DMSO is "no™
the Sso ~£ several organic compounds. Also, it should be emphasized that
the DMSO extract should never be used for chemical analyses.
proper c!rf ^in^h partlcujate samPles and ^tracts should be handled with
minded ^h .'-I y ^°ntaln 6lther mutagens °r carcinogens. It is recom-
Tdi^o^hf f I Personnel working with these materials wear proper gloves
exSctiL^ X T81",1 !10VeS' Pharma Seal ^oratories) anS carry out all
extraction steps under a laboratory hood (A-type) .
9.0 Calibration
No specific calibration procedure is required for the air matrix.
10.0 Quality Control
10.1 Sample Extraction and Concentration-
Field blanks are extracted and carried through extraction procedures
concurrently with the sample. If the field blank gives more than Jhe Jac"
t8hre°UAmesebertantS (85rtl«lc«"y -i^ic-iit compared to s-olvSfbSjkJrJn
the 5!?d biaSry; a^ sampling and field blanks have to be repeated until
the field blank gives no significant positive response in the bioassay.
11.0 Procedure
11.1 Introduction —
eenin-frv^ "^l^ f al'1(1978) Polnt out' a major consideration in the muta-
rh^o T tef ing °f ComPlex ^emical mixtures is that such mixtures contain
tJons wMS h a Vari??y °f ClaSS6S and an e^Ually wlde ra"8e °f concentra-
svneL M 3re P°tentially CaPable of "»•!»* additive, antagonistic or
synergistic responses in test organisms or at specific receptor sites. Such
interactions may be a function of the combined dose of toxicants or of the
inherent genetic susceptibility of a particular target organism. The prepara-
tion of complex mixtures for in vitro bioassay (e.g., Ames test) is compli-
cated by at least two factors: (1) the relevance of the material applied to
the test system and (2) the compatibility of the material with the test system
56
-------
(Guerin et al. 1978). Chemical "relevance" is achieved when the test system
is dosed with a material whose chemical composition mimics that which reaches
the natural point of impact. Often, difficulties with "compatibility are
encountered when the material being bioassayed contains constituents which
interfere with the test organisms' ability to respond to the effect of in-
terest. High concentrations of mildly toxic constituents or small quantities
of highly toxic constituents can sometimes mask the more subtle effect of
mutagenic constituents. Any steps taken to remove toxic constituents in order
to make the material compatible with the test system necessarily involve a
change in the physical or chemical nature of the test material. Thus, the
objective of selecting the best sample preparation protocol is to choose a
procedure which prepares materials in a form suitable,for biotesting with a
minimal or at least interpretable impact on the relevance of the test material.
The method described in this procedure has been written using a
standard ambient high-volume sample as a specific example. With suitable
modification, this procedure is applicable to most other particulate sampling '
techniques. Modifications to these procedures may be necessitated if the
sampler type or source characteristics are different from those encountered
when high-volume samples are used to collect ambient particulates. These
changes will usually involve modifications to handle a variety of sample sizes
or particulate types.
A number of methods are available for the desorption of the collected
air particulates from the high-volume filter. The three methods most commonly
used are (1) extraction, (2) thermal desorption and (3) vacuum sublimation.
Of these three, the most efficient and widely used method is the extraction
technique. In the extraction method, collected air particulates are desorbed
from the filtering medium by means of solvent(s). The extraction procedure
can be subdivided into three steps: (1) solvent extraction, (2) solvent
evaporation and (3) solvent exchange with DMSO for bioassay.
11.2 Solvent Extraction—
The principle behind solvent extraction is to dissolve the collected
particulates from the filtering medium by digesting in a suitable solvent.
The usual method involves refluxing of the filter in a Soxhlet extractor for a
certain length of time. As discussed in the Review of the Literature section,
a wide variety of solvents have been used, including acetone, benzene, cyclo-
hexane, dichloromethane, chloroform, methanol, pentane, carbon disulfide and
tetrahydrofuran. Of these, dichloromethane, cyclohexane and methanol are the
highly recommended solvents. Either extraction by cyclohexane or dichloro-
methane alone or sequential extraction using cyclohexane followed by dichloro-
methane has been recommended. The third choice is extraction using a mixture
of cyclohexane:dichloromethane:methanol (1:1:1 v/v). However, EPA recommends
extraction using either cyclohexane or dichloromethane alone (Lentzen et al.
1978, Brusick and Young 1981). The solvent extraction procedure is described
using dichloromethane as the solvent of choice. A summary flow scheme for the
procedure was previously presented in Figure 4.
Note; It is essential that a choice between these solvent systems be
made only~~after comparing the extraction efficiencies on a particular air
particulate sample. Thus, a side-by-side evaluation is recommended.
57
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11.3 Solvent Extraction Procedure—
or a soJ^r""10" "" "* d°°e U8ln " * <»t« ««•«« «*•« 7)
16 hr U 10 ™ r /£ V u extracted wlth dlchloromethane (100 tnL) for
roniJ ^ cycles/hr) at the boiling point of the solvent (40 C) . After
filtered throuLraCtOS \° *"% te">P«*ture, the two extracts are cabined and
flask (SoSmU ?h T Flu°r°P°re fllter Ott.lliporq) into a round-bottom
^ — •
^
Procedure has been evaluated by Sawicki et al.
98 27 A c K by the Sonicatlon m^hod was found to be 95% to
98.2%. A comparison of the recovery of aromatic compounds between the
sonication and Soxhlet method showed that the ultrasonic methTextracted 49%
of the total particulars matter collected on glass-fiber filters, with a
relative standard deviation ±1.33% compared to 30% for the Soxhiet extrac-
tion, with a relative standard deviation ± 26.1% of the total extractables
Son±T\?* extrac'ion efficiency but also the reproducibility of the
sonic method was superior to the Soxhlet method.
(Alfhei»HSSVTe?'/ren?oLltenaturC reconmends both these techniques equally
(Alfheim and Lindskog 1984, Hughes et al. 1980, Krishna et al. 1983b>;
11.4 Solvent Evaporation —
nart-^ i1! W±U USUa1lly be ^cessary to concentrate the extract of air
rec^nf r,K° 3 V°1Ume °f 10 mL °r 16SS for ^sequent analysis. It is
IT, T?? ^ , , concentration to slightly less than a 10-mL volume be
ban Snv± "l °8 J KudernrDanlSh (K-D) aPP^tus (Figure 8) with a three-
ereater thL ? r0 tOtal V°1UmeS betW6en 10° mL and l L« For vol«^s
greater than 1 L, a rotary evaporator (operated at 30 to 35 C, using tap water
beP,^ f0VaCT ^ circulati«8 i« water through the condenser) should
extr^t i° r^UC\the lnltlal V°lume t0 aPP^i««ately 100 mL. The resulting
material L?r c»c«t«t-dfcf«rth« b? **• I» order to prevent loss of
material, it is essential that the extract not be reduced to dryness at this
point in the preparation scheme (Brusick and Young 1981, LentJn et al!
58
-------
K-585250
Figure 7. Soxhlet extractor.
59
-------
K-570025
JL
K-570050
Figure 8. Kuderna-Danish evaporative concentrator.
60
-------
An aliquot (up to 50% or 5 mL) of this extract should be set aside (in
an amber-colored Teflon screw-capped bottle at -20 C) for mass determination
(by gravimetric analysis) and chemical analyses or for retesting in the
bioassay.
The remaining sample should then be transferred to a graduated con-
tainer (e.g., K-D receiver or centrifuge type) and concentrated to a minimum
of 1 mL or to the point where material begins to drop out of solution. In the
latter case, the extract should be restored to a convenient volume in which
the material is redissolved.
11.5 Solvent Exchange with DMSO— ,
Bioassay testing requires that the dichloromethane solvent be elimin-
ated (reduced to less than 1%) before the sample extract is applied to the
test system. An appropriate amount of extract is carefully reduced to 1 mL at
40 C under a gentle stream of purified nitrogen. The solvent evaporates
rapidly, so it is important that this operation be done under constant surveil-'
lance to ensure that the volume is not reduced below 1 mL. It is also neces-
sary to warm the samples slightly, preferably in a water bath (40 C), to
prevent condensation of atmospheric moisture in the sample caused by evapora-
tive cooling.
The sample container is held between 35-40 C in a waterbath. One
milliliter of DMSO is added and mixed by gentle agitation. The volume is
reduced to a total of slightly more than 1 mL by bubbling purified nitrogen
through the sample using a clean Pasteur pipet. Another 1 mL of DMSO is added
and mixed, and the volume is reduced to approximately 2 mL. The sample is
checked for residual methylene chloride (Brusick and Young, 1981, Lentzen et
al. 1978), and nitrogen purging is continued until the residual methylene
chloride is below 1%. The final volume of this DMSO solution is noted. The
sample may then be diluted to the appropriate concentration (1 to 10 mg/mL)
for Ames testing.
Note; In the above description steps, it is recommended that a com-
parison be~made between with/without K-D and with/without solvent exchange
steps.
11.6 Mass Determination by Gravimetric (GRAV) Analysis—
The gravimetric analysis is used for mass determination of extract-
ables with boiling points higher than 300 C. This analysis should be done
after the sample extract has been concentrated, since weighing at least 10 mg
of sample is recommended in a gravimetric analysis, when possible. Weighing
to a precision of ± 0.1 mg is adequate for purposes of Level 1 analysis.
Sample and tare weights should be obtained by drying to "constant weight"
(±0.1 mg) in a desiccator over silica gel or Drierite. In performing a
gravimetric analysis on a large volume sample (i.e., >50 mL), no more than
5 mL of extract should be evaporated to dryness. For extracts concentrated to
10 mL, a 1-mL aliquot is taken for GRAV analysis. The GRAV results should be
reported as one number for the entire sample (Lentzen et al. 1978).
61
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12 -0 Calculations
.«.«.1ct.r <£ro: say °r •
steps: accena (e.g., TA98), one can calculate the following in
a. Net revertants/volume of DMSO extract
b. Net revertants/mass of material in the total volume of the extract
c. Net revertants/total mass of particulate taken for extraction
d. Net revertants/volume of air collected in 24 hours
±S deflned as the -utagenic
12.2 Illustration—
a. Amount of total suspended particulate (TSP) =
2,000 m x M. . 2,00o ,g = 200 mg
m
b. Amount of extractable organics (assuming 10%) = 20 mg
c. Assuming a typical value of 2 revertants/yg:
2 rev./ug = 2,000 rev./mg
or = 40,000 rev. /20 mg
or = 40,000 rev./2000 m3 of air
or = 20 rev./m of air
13 -° Precision and Accuracy
62
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REFERENCES
Air Quality Criteria for Particulate Matter, National Air Pollution Control
Administration, Publication Number AP-49, January 1969.
Alfheim I, Lindskog A. 1984. A comparison between different high volume
sampling systems for collecting ambient airborne particles for mutagenicity
testing and for analysis of organic compounds. Sci. Total Environ. 34:203-
222.
Bertsch W, Chang RC, Zlatkis A. 1974. The determination of organic volatiles
in air pollution studies: Characterization of profiles. J. Chromatogr. Sci.
12(4):175-182.
Brusick DJ, Young RR. 1981. IERL-RTP Procedures Manual: level 1 environ-
mental assessment biological tests. EPA-600/ 8-81-024, U. S. Environmental
Protection Agency, Office of Research and Development, Washington, D.C.
Butler JD. 1975. Air pollution, smoking and lung cancer. Chem. Br. 11(10):
358-363.
Clements HA, McMullen TB, Thompson RJ, Akland GG. 1972. Reproducibility of
the Hi-Vol sampling method under field conditions. J. Air Poll. Cont. Assoc.
22:955-958.
Commoner B, Madyastha P, Bronsdon A, Vithayathil AJ. 1978. Environmental
mutagens in urban air particulates. J. Toxicol. Environ. Health 4:59-77.
Corn M, Montgomery TL, Esmen NA. 1971. Suspended particulate matter:
Seasonal variation in specific surface areas and densities. Environ. Sci.
Technol. 5: 155-158.
DeWiest F, Rondia D. 1976. On the validity of determinations of benzo(a)-
pyrene in airborne particles in the summer months. Atmos. Environ. 10(6):
487-489.
Dzubay RG, Stevens RK. 1975. Ambient air analysis with dichotomous sampler
and X-ray fluorescence spectrometer. Environ. Sci. Technol. 9(7):663-668.
Federal Register. 1971. No. 84.36, pp. 8191-8194.
Fennelly PF. 1975. Primary and secondary particulates as pollutants. J. Air
Pollut. Control Assoc. 25(7):697-704.
Fisher G, Crisp C, Raabe 0. 1979. Physical factors affecting the muta-
genicity of fly ash from a coal fired power plant. Science 204:879-881.
63
-------
mtin' f" 'i?1"! JN Jr' 1983' An Ultrahi8h volum* -ampler for the
multiple filter collection of respirable particulate matter. J. Air Poll
Cont. Assoc. 33:877-879.
Flessel P Guirguis G Cheng J, Chang K, Hahn E, Chan R, Ondo J, Fenske R,
Twiss S, Vance W, Wesolowski J. 1984. Monitoring of mutagens and carcinogens
in community air. Final report on the contract No. ARE Al-029-32, Review
Division, California Air Resources Board, Sacramento, CA.
Fox MA, Staley SW. 1976. Determination of polycyclic aromatic hydrocarbons
in atmospheric particulate matter by high pressure liquid chromatography
coupled with fluorescence techniques. Anal. Chem. 48(7):992-998.
Golden C Sawicki E. 1975. Ultrasonic extraction of total particulate
hydrocarbons from airborne particles at room temperature. Int. J. Environ.
Anal. Cnem. 4:9-23.
Guerin MB, Clark BR, Ho C-H, Epler JL, Rao TK. 1978. Short-term bioassay of
complex organic mixtures: Part I, Chemistry. In: Waters MD et al. (eds.),
Application of short-term bioassays in.the fractionation and analysis of
complex environmental mixtures. New York: Plenum Press, pp. 247-268.
Henry WM Mitchell RI. 1978. Development of a large sampler collector of
respirable matter EPA-600/4-78-009, U. S. Environmental Protection Agency,
Office of Research and Development, Washington, DC.
Hughes TU, Pellizzari E, Little L, Sparacino C, Kolber A. 1980. Ambient air
pollutants: collection, chemical characterization and mutagenicity testing.
Huisingh J Bradow R, Jungers R, Claxton L, Zweidinger R, Tejada S, Bumgarner
J, Fuffield F, Waters M, Summon V, Hare C, Rodriguez C, Snow L 1978
Application of short-term bioassays to the characterization of diesel'particle
emissions. In: Waters, MD et al. (eds.), Application of short-term bioassays
in the fractionation and analysis of complex environmental mixtures. New
York: Plenum Press, pp. 381-418.
Jutz GA, Foster KE. 1967. Recommended standard method of atmospheric
sampling of fine particulate matter by filter media-high-volume sampler. J.
Air Poll. Cont. Assoc. 17:1.
Kolber AR, Hughes TU, Wolff TU, Little UW, Sparacino CM, Pellizzari ED. 1983.
Development and assessment of procedures for collection, chemical characteri-
zation and mutagenicity testing of ambient air. EPA-600/S2-83-045, U S
Environmental Protection Agency, Health Effects Research Laboratory, Research
Triangle Park, NC. "
Krishna G, Nath J, Whong W-Z, Ong T. 1983b. Mutagenicity studies of ambient
airborne particles. II. Comparison of extraction methods. Mutat. Res. 124:
121—128.
64
-------
Krstulovic AM, Rosie DM, Brown PR. 1977. Distribution of some atmospheric
polynuclear aromatic hydrocarbons. Amer. Lab., July, pp. 11-1B.
Lentzen BE. Wagoner DE, Estes ED, Gutknecht 1978 ^f^^^0^"^.^!
Manual: Level 1 Environmental Assessment (Second Edition). EPA-600/7-78 201,
U. S. Environmental Protection Agency, Office of Research and Development,
Washington, DC.
Lippmann M, Albert R, Yeaters D. 1979. Effects of inhaled particles on human
and animals: deposition, retention and clearance. In: Air particulates.
National Research Council. Baltimore: University Park Press, pp. 107-145.
\
Natusch D, Wallace J. 1974. Urban aerosol toxicity: the influence of
particle size. Science 186:695-699.
Ogden TL, Wood JD. 1975. Effects of wind on the dust and benzene-soluble
matter captured by a small sampler. Ann. Occup. Hyg. 17:187-195.
Pierce RC and Katz M. 1975. Determination of atmospheric isomeric polycyclic
arenes by thin-layer chromatography and fluorescence spectrophotometry. Anal.
Chem. 47:1743-1748.
Pitts JN, Jr. 1983. Formation and fate of gaseous and particulate mutagens
and carcinogens in real and simulated abmospheres. Environ. Health Perspect.
47:115-140.
Pitts JN, Jr., Van Caumenberge K, Grosjean D, Schmid J, Fits D. Belser W,
Knudson S, Hynds D. 1978. Atmospheric reactions of polycyclic aromatic
hydrocarbons: Facile formation of mutagenic nitroderivatives. Science.
515-519.
Preidecker BL. 1980. Comparative extraction of Houston air particulates with
cyciohexane or a mixtue of benzene, methanol and dichloromethane II. Envxron.
Mutagen. 2:85-88.
Pupp C, Lao RC, Murray JJ, Pottle RF. 1974. Equilibrium vapor concentrations
of some polycyclic aromatic hydro-carbons, AS 0, and S 02 and the collection
efficiencies of these air pollutants. Atmosp. Environ. 8:915-925.
Schlesinger R, Lippmann M. 1978. Selective particle deposition and broncho-
genie carcinoma. Environ. Res. 15:424-431.
Schuetzle D, Crittenden AL, Charlson RJ. 1973. Application of computer
controlled high resolution mass spectrometry to the analysis of air
pollutants. J. Air Pollut. Control Assoc. 23(8):704-709.
Sholtes RS, Engdahl RB, Herrick RA, Phillips C, Stein E, Wagman J, Woolrich
PR. 1970. Tentative method of analysis of suspended particulate matter in
the atmosphere (high volume method). Hlth. Lab. Sci. 7:279-286.
Sweetman JA, Harger W, Fitz DR, Paur H-R, Winer AM, Pitts JN Jr. 1984.
Diurnal mutagenicity of airborne particulate organic matter adjacent to a
65
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66
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SECTION 3
DRINKING WATER
REVIEW OF THE LITERATURE
Introduction
The presence of trace levels of organic compounds in drinking water has
heightened the concern among the scientific community and throughout the
public sector as to the possible human health impact of chronic exposure to
these contaminants. An initial response to these concerns was the enactment
of a series of Federal Acts, including the Safe Drinking Water Act of 1974, PL
93-523, and the Clean Water Act of 1977, PL 95-217. Although the implemen-
tation of this federal legislation has resulted in measurable improvements in
the nation's drinking water in terms of the removal of toxic metals and the
lowering of the concentrations of some organics such as trihalomethanes (CEQ
1979, 1980, 1981, 1982), "further studies of the identities, carcinogenicity,
mutagenicity, mode of formation and practical methods of removal are needed
for the organic contaminants in drinking water" (Crump and Guess 1980). A
great variety of natural and synthetic chemicals find their way into both the
surface and ground waters used as sources of drinking water. The chemical/
physical properties of these compounds have allowed for measurable progress in
some areas of improved water quality, e.g., detection and subsequent lowering
of trihalomethanes, but there has been a lack of progress in other areas,
e.g., isolation/identification and subsequent removal of mutagenic
constituents in the nonvolatile residue organics.
In terms of chemical analysis, this broad spectrum of organic chemicals
in water is divided into two categories: (1) compounds with low solubility
and a volatility sufficient for rapid separation and identification by gas
chromatography/mass spectrometry and (2) relatively soluble compounds with low
volatility, which are not readily separated by purge/extraction techniques,
but are collected by more extensive extraction/concentration techniques from
water as complex mixtures of residue organics. These two categories contain
approximately 10% and 90%, respectively, of the organics in drinking water
(Garrison 1977). As noted in a U.S. National Academy of Sciences - National
Research Council Report (NAS-NRC 1977), all but about 10% of the organic
compounds in the first category have been identified and can be quantitated.
This progress has been facilitated by the development of methods to isolate
and quantitate these compounds, as discussed in the following selected reviews
(Smith 1978, Nunez et al. 1984), compendia (Keith 1976, 1981) and detailed
procedures (44 FR 69464, December 3, 1979, Coleman et al. 1981). Some of the
identified organics in this category have been assessed as to their toxico-
logical significance (NAS-NRC 1977, 1980a, b, 1982a, 1983), and the evaluation
67
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Overview of Procedures for Isolating Residue Or^anics
.
great majority of these compounds are unknown and are present in onlv dllut*
solution, it is impossible to test the absolute efficient any one method.
«'
£^^
nonpolar stationary phases. Different combinations of many of these methods
a6 "" 1S°latiOn °
water in order tn 1S°lation °f nonvolatile residue organics from drinking
water in order to improve recoveries or process large sample volumes.
Methods Based on Water Removal—
The preparation of aqueous concentrates via water removal is limited by
solubil^fnf^11 in°r8anic s^stances in the sample and the aqueous
specie organic substances. As the water is removed, the inorganic
P are concentrated along with the organics. Following the noinf of
re overv oTthT^' ?* ^T^ "* "^ Sp6CleS -pre^ipita'te^'making
recoverv of the organic constituents of interest difficult. However, aqueous
concentrated to a point nearing saturation can be used directly for
'Li J! TSter Sy!tem ^3S ade
-------
where the salts interfere and/or the solution is still too dilute, the con-
centrates must be processed further by alternative methods such as extraction
or chromatography. In general, the methods of freeze-concentration (Shapiro
1961, Malo 1967a, b, Baker 1970), freeze-drying or lyophilization (Dawson and
Mopper 1978, Crathorne et al. 1979) and evaporation or distillation under
reduced pressure (Jolley et al. 1975) have not been used for the preparation
of residue organics from drinking water for bioassay due to the aforementioned
limitations. However, these methods have been used for the preparation of
residue organics from drinking water for chemical analysis (for example, see
Buelow et al. 1973, Watta et al. 1982).
The concentration method of reverse osmosis has bveen used in the prepara-
tion of drinking water residue organics for bioassays. Although this method
was originally reported by Hindin et al. (1969), further development was
conducted by Deinzer et al. (1975) in order to concentrate trace organics from
drinking water for compound identification by combined gas chromatography/
mass spectrometry techniques. Further studies of the methodology by Kopfler
et al. (1977) resulted in the development of a method that was successfully
applied to six different U.S. drinking water supplies for the preparation of
residue organics for toxicological assessment and compound identification.
The method features partition of the reverse osmosis concentrates by extrac-
tion of the organics into petroleum ether, diethyl ether and acetone. Each
reverse osmosis concentrate extract was initially shown to be mutagenic in
spot tests by Simmons and Tardiff (1976). These preliminary studies prompted
modification of the procedure to include the isolation of additional residue
organics from the remaining aqueous concentrates via XAD-2 chromatography.
The extracts and XAD-2 isolates were subsequently bioassayed in a variety of
short-term tests by Loper et al. (1978), Lang et al. (1980) and Kurzepa et al.
(1980) and shown to be mutagenic in bacterial test systems and to cause
cellular transformation in mammalian cell lines.
The method of Kopfler et al. (1977) warrants a few additional comments.
This reverse osmosis concentration system employs both cellulose acetate and
nylon membranes for the concentration of organics. The two different types of
membranes were used because of their more efficient selectivity in the rejec-
tion of specific classes of compounds: the former rejects ionized solutes and
polar constituents, particularly hydroxy compounds, whereas the latter rejects
nonpolar constituents, such as aromatic compounds. The recovery efficiency
was high for the cellulose acetate membrane (93%), but efficiencies could not
be calculated for the nylon membrane due to the low total organics concentra-
tions in the influent. The overall efficiency of this method for the organics
concentrated from the drinking waters of three of the six cities in the study
ranged from 15% to 41%. However, one problem noted with this method was the
leaching of a number of compounds from the membranes into the concentrates. A
more significant problem with the reverse osmosis method was the accumulation
of inorganic salts in the residue organics concentrate. Some organics were
adsorbed to the salts, thereby making recovery difficult.
Methods Based on Isolation of Organics from the Water—
The preparation of extracts of residue organics by isolation techniques
requires the use of solvents either in the direct liquid/liquid extraction of
the drinking water or in the desorption of chromatographic stationary phases
69
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limitationinthe-0rf^CS ^T ** ^^ ™teT' The Solvent ls the *****
limitation in the isolation techniques, in that impurities can be concentrated
along with the organics being isolated. The most common sources of solved
. common sources o solve
impurities are the preservatives, usually antioxidants, that can react with
sample components (Kopfler, 1980). Also, solvent impurities can interfere
with subsequent bioassays of the isolated nonvolatile residue organics
soeban ' S°venS a" t
solvent blanks is mandatory for all isolation techniques.
Solvent extraction techniques have had limited applications in the prepa-
Two of tLT °f8ani,CS frT drlnk±ng W3ter f°r lexicological assessment!
the mfthnd M"^ i * ^ °f Investi8ati™ of tfa*s procedure may be that
the method is highly selective and is cumbersome, since large amounts of
liquids have to be manipulated. However, the method has been investigated for
use in the removal of organics from drinking water (Suffet et al. 1976) and
has been employed to isolate organics from water for quantitation of con-
taminants (for example, Mieure and Dietrich 1973, Hites 1977). Additionally, •
it should be noted that Meier and Bishop (1984) recently reported the use of
this method for the preparation of residue organics from wastewaters for
mutagenic assessment.
The isolation technique developed most extensively is the use of acti-
vated carbon as a stationary phase in chromatography to adsorb organics from
water passed through the column. The method has been used for decades to
isolate organics from drinking water for compound identification and for water
quality monitoring (Braus et al. 1951, Middleton et al. 1956, Rosen, 1976,
McGuire and Suffet 1983) . The method has been developed to process thousands
i 8?Qfi9?S °T W3tef t0 yleld gram <*uantlties of ^sidue organics (Middleton et
lsolat
T
•v. vi * lsolate the residue organics, the activated carbon is extracted
with chloroform and ethyl alcohol, In sequence, followed by solvent removal
via evaporation. However, the recovery of organics from the activated carbon
is not as reproducible as with other methods (Chriswell et al. 1977) More
recently the use of supercritical liquid carbon dioxide has been examined to
extract the carbon (Modell et al. 1978). This extraction method may be useful
not only because liquid carbon dioxide is a good solvent for several classes
of organics, but also because the high temperatures needed for organic solvent
extraction are not required. It should be noted that a previously undescribed
potent mutagen was isolated (Tabor and Loper 1980) and identified (Tabor 1983)
from a portion of a 125-gram sample of residue organics isolated from 50,000
gallons of finished drinking water via the "Megasampler" (Middleton et al.
1962) .
Organic polymers have been used extensively as stationary phases for
adsorbing organics from drinking water. The most widely used polymers
(Kopfler 1980, Jolley 1981, NAS-NRC 1982b) are either the polystyrenedivi-
nylbenzene copolymers or the polymethacrylate polymers, both commonly referred
to as various XAD resins. The use of XAD resins for the concentration of
nonvolatile residue organics from water was first described by Burnham et al.
(W/t). In this study, weak organic acids and bases and neutral organic
compounds were adsorbed on XAD-2 and /or XAD-7 from water solutions at original
-
n*/. the Parts-per-billion to parts-per-million range, some with
about 100^ efficiency. In a series of follow-up experiments (Junk et al.
70
-------
1974), a larger number of compounds, spiked into water, were recovered from
the XAD resins at efficiencies of 80% to 100%. These laboratory studies have
been followed by other similar investigations. Malcolm et al. (1977) found
that adsorption optimization requires control of both the flow rates and the
pH of the sample. Furthermore, they estimated that an XAD-8 column could
concentrate about 50% of the total organic carbon of the average water sample.
The studies of Thurman et al. (1978, 1979) featured the use of XAD-8, a some-
what polar resin similar to XAD-7, and examined the efficiency and capacity of
this resin for the adsorption of polar organics from water. One important
result of the study was the derivation of an empirical relationship allowing
for the matching of column sizes to sample size. In a more extensive study of
XAD resins, van Rossum and Webb (1978) measured the recovery efficiencies of
XAD-2, -4, -7 and -8 for organics from water. Additionally, they evaluated a
variety of organic solvents for the elution of adsorbed compounds from the
resins. Although they recommended the use of equal amounts of XAD-4 and
XAD-8, Cheh et al. (1980) have reported that passage of water containing
residual chlorine over XAD-4 yields mutagenic artifacts. Likewise, James et
al. (1981) and Care et al. (1982) have reported the leaching of XAD resin
components when the columns were eluted to recover residue organics from
water. Therefore, it is imperative that XAD resins are cleaned extensively
prior to use. Procedures for accomplishing this have been published (Junk et
al. 1974, Webb 1975, USEPA 1978). Additionally, general studies of the
chemistry of the XAD resins and their use as high performance liquid chroma-
tography supports have been reported (Grieser and Pietrzyk 1973, Pietrzyk and
Chu 1977a, b, Pietrzyk et al. 1978, Baum et al. 1979).
The XAD resins have been used extensively for the concentration of
residue organics from drinking water for toxicological assessment. One reason
for their wide use is based on the following argument of Yamasaki and Ames
(1977). Since the XAD resins exhibit a high specificity for adsorption of
apolar or lipophilic compounds of low polarity (Junk et al. 1974, Fritz 1979,
Dressier 1979), organic compounds, which will exert biological effects and are
thus capable of passing through biological membranes, are likely to fall in
these categories. Glatz et al. (1978) and the Canadian research group (LeBel
et al. 1979, Nestmann et al. 1979, Williams et al. 1982) utilized the nonpolar
XAD-2 to prepare residue organics from drinking water for bioassay. One Dutch
research group (Van der Gaag et al. 1982, Noordsij et al. 1983) utilized a
series of three XAD-4 columns for the isolation of residue organics at pH 7, 2
and 9, respectively. Several research groups have used a combination of two
different XAD resins for the isolation of nonvolatile residue organics.
Another Dutch research group (Kool et al. 1981a, b, 1982, 1984, Zoetemann et
al. 1982) utilized a combination of XAD-4 and XAD-8, whereas one U.S. research
group (Loper et al. 1983, Loper et al. 1984, Tabor and Loper 1984) utilized a
combination of XAD-2 and XAD-7. The XAD resins have been applied to waters
other than drinking water for the isolation of residue organics for toxico-
logical assessment. For leading references see Jolley (1981), Hoffman (1982),
Maciorowski (1982) and Tabor et al. (1984).
Additional methods, employing not only XAD resins but also other station-
ary phases and/or techniques, have been investigated for use in concentrating
residue organics from water. Carbridenc and Sidka (1979) have developed a
combined solvent extraction/XAD procedure that was reported to give a total
71
-------
•
packed In distinct lasers with ?£ ^I" Orl8±nal meth°d, the column was
Recommended Procedure for Isolating Residue Organics
ing witer'ifthe" nrL!±-d f°J the is?lation of «sld«e organics from drink-
,
o?Pthe methido"™"?: g jlr'L^8'.'^!,11,0' V*' "84) and —«*"«
t-inn nf MB4j ^e g. labor et al . 1984) have been used for the prepara
tion of residue organics from wastewater for mutagenicity testing.
72
-------
A few comments on the apparatus and methodology (which are described in
the Protocol) are warranted. The system is constructed of stainless steel,
thereby providing for a durable system. All fittings are of Swagelok design,
thereby allowing for multiple reuse without leakage, etc. The column pack-
ings, resins or glass wool, are supported on both ends by stainless steel
sintered frits. The system is adaptable to large or small sized samples under
a variety of line pressure situations. Also, the use of in-line bacterial
filters allows some time between sample collection and column elution.
As to the choice of resins, preliminary experiments (Van Rossum and Webb
1978, Loper et al. 1983, 1984, Tabor and Loper 1984, Kool et al. 1981a, b,
1982) have shown that the combination of XAD-2 (or 4) and XAD-7 (or 8) will
extract nonpolar and semipolar residue organics from dilute aqueous solutions
like drinking water. Furthermore, the extracted organics are readily eluted
from the resins. Although numerous solvent systems have been utilized for the
elution of the residue organics from the XAD resins (Junk et al. 1974, Van
Rossum and Webb 1978, LeBel et al. 1979, Baird et al. 1980, 1981, Van der Gaag
1983, Noordsij et al. 1983, Nellor et al. 1984), Tabor and Loper (1984) have
shown the hexane:acetone system of LeBel et al. (1979) to be more suitable for
extracting residue organics. Other solvent systems, e.g., diethyl ether or
acetone followed by methylene chloride, gave either mutagenic artifacts or low
yields in terms of recoverable mutagenesis. This latter study (Tabor and
Loper 1984) established flow rates, sample volume limitations and general
operation parameters for the system described in the Protocol section.
The procedure described in the Protocol may not recover the highly polar
and ionic organic species along with the highly volatile, low molecular weight
organics. As to the former category of organics, further studies of the
systems of Baird (Baird et al. 1980, 1981, Jenkins et al. 1983, Nellor et al.
1984) or of the Dutch National Waterworks group (Van der Gaag et al. 1982,
Noordsij et al. 1983) may provide a suitable method. The highly volatile
organics can be quantitated via other methods (e.g., Coleman et al. 1981,
44 FR 69464, December 3, 1979). Alternatively, the system proposed by Van der
Gaag et al. (1982) allows for the collection of the volatile organics at the
same time as that of the residue organics. Although this system is not as
portable as the one described in the Protocol, it is possible that the Dutch
system will be developed further for future wide-range application and use.
73
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REFERENCES
papers'-
DC: American Chemical Society.
.
— Mng Vol. 20(1), Washington,
DC: American Chemical SocUty.
Chemistry.
meeting Vol. 24(2), Washington,
ronns cnton i ' °"ParS™ ° »»
Anal. Chem. 51(11) :1799!l803 aCld fr<"° a1»eous solution.
IV.
Science Publishe In W 9-935 '
a
*°° *"""' MI' A™ Arb<)r
macroporous resin
an.
74
-------
Burnham AK, Calder GV, Fritz JS, et al. 1972. Identification and estima-
tion of neutral organic contaminants in potable water. Anal. Chem.
44(1):139-142.
Carbridenc R, Sidka A. 1979. Extraction of organic micropollutants from
water for the purpose of performing biological assays. Presented at the
European Symposium on the Analysis of Organic Micropollutants in Water,
Berlin, Federal Republic of Germany. (In French; translation).
Care R, Morrison JD, Smith FJ. 1982. On limits of detection of traces of
volatile organics in water, using Amberlites XAD-2 resin. Water Res.
16:663-665.
Cheh AM, Skochdopole J, Koski P. 1979. Nonvolatile mutagens in drinking
water: production by chlorination and destruction by sulfite. Science
207:90-92.
Chriswell CD, Ericson RL, Junk GA et al. 1977. Comparison of macroreticular
resin and activated carbon as sorbents. J. Am. Water Works Assoc.
69:669-674.
Coleman WE, Melton RG, Slater RW et al. 1981. Determination of organic con-
taminants by the Grob closed-loop-stripping technique. J. Am. Water Works
Assoc. 73:119-125.
Council on Environmental Quality. 1979. Environmental Quality, the 10th
Annual Report of the Council on Environmental Quality. Washington, DC: US
Government Printing Office.
Council on Environmental Quality. 1980. Environmental Quality, the llth
Annual Report of the Council on Environmental Quality. Washington, DC: US
Government Printing Office.
Council on Environmental Quality. 1981. Environmental Quality, the 12th
Annual Report of the Council on Environmental Quality. Washington, DC: US
Government Printing Office.
Council on Environmental Quality. 1982. Environmental Quality, the 13th
Annual Report of the Council on Environmental Quality. Washington, DC: US
Government Printing Office.
Crathorne B, Watts CD, Fielding M. 1979. Analysis of non-volatile organic
compounds in water by high-performance liquid chromatography. J. Chromatogr.
185:671-690.
Crump KS, Guess HA. Science Research Systems, Inc. 1980. Drinking water and
cancer: Review of recent findings and assessment of risks. Prepared for
Council on Environmental Quality - Springfield, VA: National Technical
Information Service, US Department of Commerce, p. xiii. Contract No.
EQ10AC018.
75
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of solutes £rom
ln water to
°£ """S6"8 fr<» -««r by the
76
-------
Jolley RL. 1981. Concentrating organics in water for biological testing.
Environ. Sci. Technol. 15(8):874-880.
Junk GA, Richard JJ, Grieser MD. 1974. Use of macroreticular resins in the
analysis of water for trace organic contaminants. J. Chromatogr. 99:745-762.
Keith LH. 1976. Identification and analysis of organic pollutants in water.
Ann Arbor MI: Ann Arbor Science Publishers, Inc.
Keith LH. 1981. Advances in the identification and analysis of organic
pollutants in water Vol. 1 and 2. Ann Arbor, MI: Ann Arbor Science
Publishers, Inc. » *
Kool HJ, Van Kreijl CF, Van Kranen HJ et al. 1981a. The use of XAD-resins
for the detection of mutagenic activity in water. Chemosphere 10:85-98.
Kool HJ, Van Kreijl CF, Van Kranen HJ et al. 1981b. Toxicity assessment of
organic compounds in drinking water in the Netherlands. Sci. Total Environ.
18:135-153.
Kool HJ, Van Kreijl CF, Zoeteman BCJ. 1982. Toxicology assessment of organic
compounds in drinking water. CRC Critical Rev. Environ. Contr.
12(4):307-359.
Kool HJ, Van Kreijl CF, Van Oers H. 1984. Mutagenic activity in drinking
water in the Netherlands. A survey and a correlation study. Toxicol.
Environ. Chetn. 7:111-129.
Kopfler FC, Coleman WE, Melton RG et al. 1977. Extraction and identification
of organic micropollutants: Reverse osmosis method. Ann. NY Acad. Sci.
298:20-30.
Kopfler FC. 1980. Alternative strategies and methods for concentrating che-
micals from water. In: Waters MD, Sandhu SS, Hueisingh JL, Claxton L, Nesnow
S, eds. Short-Term Bioassays in the Analysis of Complex Environmental
Mixtures II. New York: Plenum Publishing Corp., pp 141-153.
Kurzepa H, Lang DR, Kyriazis A. 1980. Tumor transformation and latency
period by chemically transformed and morphologically normal BALB/3T3 cells in
athymic mice. Proc. 31st Ann. Meeting Tissue Culture Association, June, St.
Louis, MO.
Lang DR, Kurzepa H, Cole MS et al. 1980. Malignant transformation of
BALB/3T3 cells by residue organic mixtures from drinking water. J. Pathol.
Exp. Toxicol. 4:41-54.
LeBel GL, Williams DT, Griffith G, Benoit FM. 1979. Isolation and con-
centration of organophosphorus pesticides from drinking water at the mg/L
level. J. Assoc. Off. Anal. Chem. 62(2):241-249.
77
-------
water.
sr?r-
car.
1956' Drlntln8
Chen. Eng
of
-
If
78
-------
NAS-NRC. 1980a. Drinking water and health. Vol. 2. Report on the safe
drinking water committee, Board of Toxicology and Environmental Health
Hazards, Assembly of Life Sciences. Washington, DC: National Academy Press.
NAS-NRC. 1980b. Drinking water and health. Vol. 3. Report on the safe
drinking water committee, Board of Toxicology and Environmental Health
Hazards, Assembly of Life Sciences, Washington, DC: National Academy Press.
NAS-NRC. 1982a. Drinking water and health. Vol. 4. Report on the safe
drinking water committee, Board of Toxicology and Environmental Health
Hazards, Assembly of Life Sciences. Washington, DC: National Academy Press.
\
NAS-NRC. 1982b. Qualify criteria for water reuse. Panel on quality criteria
for water reuse, Board on Toxicology and Environmental Health Hazards
Commission on Life Sciences, Washington, DC: National Academy Press.
NAS-NRC. 1983. Drinking water and health. Vol. 5. Report on the safe
drinking water committee, Board of Toxicology and Environmental Health
Hazards, Assembly of Life Sciences, Washington, DC: National Academy Press.
Nellor MH, Baird RB, Smyth JR. 1984. County Sanitation Districts of Los
Angeles County. Health effects study. Final report. Whittfer, CA: County
Sanitation Districts of Los Angeles County.
Nestmann ER, LeBel GL, Williams DT, Kowbel DJ. 1979. Mutagenicity of organic
extracts from Canadian drinking water in the SaImone11a/Mammalian microsome
assay. Environ. Mutagen. 1:337-345.
Noordsij A, Van Beveren J, Brandt A. 1983. Isolation of organic compounds
from water for chemical analysis and toxicological testing. Intern. J.
Environ. Anal. Chem. 13:205-217.
Nunez AJ, Gonzalez LF, Janak J. 1984. Pre-concentration of headspace vola-
tiles for trace organic analysis by gas chromatography. J. Chromatogr.
300:127-162.
Pietrzyk DJ, Chu C. 1977a, Amberlites XAD copolymers in reversed phase gra-
vity flow and high pressure liquid chromatography. Anal. Chem. 49(6):757-764,
Pietrzyk DJ, Chu C. 1977b. Separation of organic acids on Amberlite XAD
copolymers by reversed high pressure liquid chromatography. Anal. Chem.
49(6):860-866.
Pietrzyk DJ, Kroeff EP, Rotch TD. 1978. Effect of solute ionization on
chromatographic retention on porous polystyrene copolymers. Anal. Chem.
50(3):497-501.
Rosen AA. 1976. The foundations of organic pollutant analysis. In: L.H.
Keith (ed.). Identification and analysis of organic pollutants in water, Ann
Arbor, MI: Ann Arbor Publishers, pp. 3-14.
79
-------
Science 133:
"** "****>* for concentration of dilute
Simmons VF, Tardlff T?r
cantratas. Mutat Res."
°f
water oon-
Tabor MW, Loper JC, Myers BL, Rosenblum L, Daniels FB
o, COBP
1QRA
Chemical Analyst, of
Protection A8Ly,°pp 1-5.
M"h°d 33°'2 ln Methods for
"' *'
80
-------
Van Der Gaag MA, Noordsij A., Poels CM, Schippers JC. 1982. Orienterend
onderzoek met analytlsch-chetnische en genotoxicologische meetmethoden naar het
effect rian waterbehandelingsprocessen. H20 15:539-558 (In Dutch).
Van Rossum P, Webb RG. 1978. Isolation of organic water pollutants by XAD
resins and carbon. J. Chromatogr. 150:381-392.
Watts CD, Crathorns B, Fielding M et al. 1982. Nonvolatile organic compounds
in treated waters. Environ. Hlth. Perspect. 46:87-99.
Webb RG. 1975. U. S. Environmental Protection Agency. Southeast Environ-
mental Research Laboratory. Isolating organic water pollutants: XAD resins,
urethane foams, solvent extraction. Final report. Springfield, VA: NTIS,
U. S. Department of Commerce, EPA-660/4-75-003. Accession No. PB-245 647.
Williams DT, Nestmann ER, LeBell GL, Benoit FM, Otson R. 1982. Determination
of mutagenic potential and organic contaminants of Great Lakes drinking water.-
Chemosphere. 11(3):263-276.
Yamaski E, Ames BN. 1977. Concentration of mutagens from urine by adsorption
with the non-polar resin XAD-2: Cigarette smokers have mutagenic urine.
Proc. Natl. Acad. Sci. U.S.A. 74:3555-3559.
Zoeteman BDG, Hrubec J, deGreed E, Kool HJ. 1982. Mutagenic activity
associated with by-products of drinking water disinfection by chlorine,
chlorine dioxide, oxone and UV-Irradiation. Environ. Hlth. Perspec.
46:197-205.
81
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DRINKING WATER WORKGROUP REPORT
Workgroup Tasks
Primary Tasks —
employing the protocol ^HM?V written into any experimental design
rA m°d"icatlon to th- P^tocol was to change the flow
ratesoe ow
flow rtes of So S%?0 T / ^"/fS10 thr°Ugh the 8?St**' Originally,
82
-------
Specific study requirements—Several questions and issues were raised in
the general sessions that the Drinking Water Workgroup decided could be dealt
with more appropriately as requirements of a specific study in which the
protocol is employed. One question concerned the frequency of sampling for a
given drinking water, and another concerned the number of parallel samples to
be collected. The statistical design of a particular study requiring the use
of the protocol will determine the frequency and number of samples needed to
accomplish the objectives of a given project. However, it was recommended
that a duplicate set of samples be taken as a minimum sample set in order to
provide for cases where something may happen to one sample set.
Additional research needs—One issue not addressed by the protocol was
the use of spikes and/or surrogates to assess operation efficiency for a
particular drinking water sampling. A series of compounds needs to be chosen
and evaluated for use as spikes and/or surrogates in the protocol.
A second related research need for this protocol is to characterize the
system more completely as to recoveries of compounds from drinking water.
Compounds used for this study should encompass a range of solubilities and
chromatographic retention indices.
Due to the limitations of the XAD resins in terms of the types of
organics (e.g., nonpolar versus polar) absorbed from dilute aqueous solution,
additional investigations on the use of additional columns, e.g., MP-1 or
MP-50, following the XAD columns are warranted.
Secondary Tasks—
A number of other questions were raised by the general group as to the
use of this protocol. These tasks, assigned to the drinking water group, were
discussed and the results are summarized below.
Type of water sample—Several questions were raised as to the type of
water sample used in this protocol for the concentration of residue organics.
In general, all types of waters, containing <5% solids, can be used as samples
for this protocol. However, the inclusion of a 25-cc bed volume column of
Celite 545 in the system between the glass wool prefilter column and the
bacterial filters is recommended for waters having a total organic carbon
level greater than 20 ppm or containing particulate matter. A discussion of
the inclusion of this column for wastewaters is contained in Tabor et al.
(1984) and has been added to the protocol.
As to the type of disinfection used for drinking water treatment or to
the source (ground versus surface) of drinking water, the protocol is viable
for all of these types of finished drinking water.
Other issues from discussion of the air sampling protocol—A number of
questions were raised during the discussion of the air sampling protocol that
were to be considered by the drinking water group. The issues included: Vola-
tile organics, solvent extraction, input requirements, definition of blanks,
storage of samples, internal standards, solvent volume reduction, solvent
83
-------
summary.
sections of this sugary
Key Peer Review Comments—
1SSUeS
in other sections of this
i» the general group
Were d±scu*^ ±J other
discuss the applicability Ind
types of residue orgMlJ
been addressed In both
o
pr°t°c°1
""" "
Potocol In terms of the
or .
limitations of the procedure. Tni°
.1 research needs for this protocol
""- a Action
pr°tOCo1 and the
. with regard to
These issues wer «.ol™d inthe drlnkT^ ?"*" °f el"tlt"1 «<>l"i".
appropriate changes were Ide to the protocS!" 8r<""> dlsCUS8l°"=. -^
Summary of Drinking Water Borkgrou[> Progres8..
were discuss* FJZ^Z™'** °^™ <>f this report
"
its
residue organics from dri.
Summary of Key Workfirm,p Dlscusslona
»ere outlined in the
Major Consensus Opinions—
sections. ±SSU6S/taSkS dlSCUSS6d
reso1^ as noted in the preceding
84
-------
Unresolvable Issues—
There were no unresolvable issues in the drinking water workgroup.
Evaluation of Proposed Protocol
The protocol for the preparation of residue organics from drinking water
for mutagenicity testing is the result of a number of investigations by
various research groups in both North America and Europe. This protocol
adequately addresses the task assigned to the drinking water group in pro-
viding a proven method for the isolation of residue organics from drinking
water for mutagenicity testing. The method is applicable to the isolation of
residue organics from drinking water purified from both surface and ground
sources. The method provides for the reproducible qualitative recovery of
residue organics from drinking water. The procedure may not recover the
highly polar and ionic species along with the highly volatile, low molecular
weight organics. With the inclusion of a 25-cc bed volume column of Celite
545, the protocol is applicable to wastewaters containing 5% solids. The
method is restricted to use by, or under the supervision of, analysts experi-
enced in chromatography and properly trained in the handling and use of
biohazardous materials.
The protocol does not apply to those mutagens not absorbed on or not
desorbed from, the XAD resins and other system components, e.g., glass wool.
It is not applicable to the more volatile organics or to highly unstable muta-
gens which may be lost/destroyed/modified during the sampling or work-up
procedures. It is not known what percentage of the total "mutagenicity" of a
water sample is recovered by this protocol. However, data for multiple
parallel samplings of a drinking water (Tabor and Loper 1984) indicate the
precision of the method to be within the limits of the Ames test for the
residue organics. There are some data (e.g., Kool et al. 1982a, 1984, Tabor
and Loper 1984) to indicate that mutagenic artifacts are not formed by the
chlorine residual in drinking water reacting with the system components (e.g.,
resins), but there is almost no data for other disinfectants which could be
used (i.e., ozone, etc.) (Kool et al. 1981a, 1984, Zoetemann et al. 1982).
The bioassay of the drinking water residue organics does not differentiate
between mutagens present in the raw water and those formed during water treat-
ment for the preparation of finished drinking water. The raw water data can
be obtained via application of the wastewater protocol to the raw water.
In comparison to the five other media protocols, this protocol is
applicable to aqueous samples derived from the wastewater protocol but is not
applicable to the other four protocols.
The drinking water workgroup strongly and unanimously recommended that
all workgroup reports be included as a separate review section in the final
document of protocols. This is imperative, since these reports will present
to the user not only the evaluations of the protocols but also the limitations
and research needs for the protocols.
85
-------
Other Data or Information Requirements
Information Gaps —
Research Program Needs —
of the compounds through all components used in the protocol? aCCOUntabillty
im-l t? addj"onal asPect to be considered in these studies would be the
inclusion of known mutagenic compounds or compounds having structures
™'~"'
«.,,r,.;»;:,2 :?,Z.-:»S.:;.T
which to includ! !*Chan8YeSinS should ^ investigated. The logical Join? at
d
wc to includ .
nce the ^ d i columns would be following the XAD resins columns,
since the XAD columns remove the nonpolar and semipolar organics. Therefore
the ion
empoar organcs. Therefo
the ion exchange columns would receive an influent water containing polar
0168' ^^ StUdleS Sh°Uld include the us^ °f ««x»el compounds to
recoveries, as previously described under "Recovery studies?"
system0descr?bSed0^Uth "' "^ "P^""-The current data on the use of the
system described in the protocol suggest that the method provides for the
86
-------
reproducible qualitative recovery of mutagenic residue organics from drinking
water. However, the development of an adequate quality assurance plan for
this protocol will require the inclusion of steps whereby recoveries and
system performance from sample to sample can be monitored; this can be accom-
plished by spiking the sample with known compounds. The choice of compounds
is open to discussion at this time. Part of the answer as to the type of
compound(s) will come from the results of the research previously suggested
under "Information Gaps." However, compounds representative of known mutagens
should be considered.
87
-------
PROTOCOL FOB THE PREPARATION OF »KlmiK VKm FOE
1>0 Scope and Application
water intended for human consumption.
water ls defined as
appllcable „ other waters (g.
organics are defined in Section 3.2.
water. Residue
P^r
samples to the laboratory
r6qulre the transport of water
ror
and use of biohazardous material!
2.0 Summary of Methods
mers and polymethacrvlate
sample th?ougT th ^columns
tion system components with
evaporation and
nitrogen until
the handlln8
rter is passed
lystyrenedivlnylbenzene copoly-
?****' follow^8 P«B.geVthe
Or8anlcs are eluted from the collec-
the «olv«t. are removed b
2.2 The
al. (1979), Loper"eral"*?1983U ^'fiA/^^01 ±S baSed °n reP°rts ^7 LeBel _.
(1981), Jenkins et al. ^983) and ^^^1*^198^ ll^tl "f11? ** **'
residue organics from drinking water Jnri-J I isolation of
due organics are those which frl«£' * JV purposes of ^is method, resi-
described herein and recovered by the BolJ^f."?1"? "^^ the cond±tlons
The procedure may not recover the hilhlv f * meth°d °f
- highly volatile, low molecular wXght^rganicl ^ T^
* u coign aZlfl 16St ITlR Of
88
-------
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 organics. Additionally, the
passage of chlorinated, 2-ppm, ASTM Type I water through the system did not
produce residue organics 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
organics 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 ymho/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 organics 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 organics eluted from the resins following passage of
ASTM Type I water through the assembled collection system.
3.6 Residue Organics—
Those organics adsorbed by XAD resins under the conditions described
herein and recovered by the solvent elution method of this protocol.
3.7 Solvent Blank—
Elution solvents are concentrated 1,000 times, and the concentrates
are bioassayed for mutagenesis.
89
-------
4-0
Interferences
it
vary fromnsuprpfUeTto- S%pUeranTf1C"
recommended that pe«l'Xe-«ad« or f™ 8'? " f™"8' Ih"e£°". it is
appropriate tests of "a" ill of 111?"? -J1'™'" •« «««! and that
cedures dascrlbed in lection 9?1. '" Conducted ^cording to the pro
t
its equivalent, described in Section 6 1? fta
come into contact only with " ° 8
«.ll. and utiH2in8 only
chrcai
s«el apparatus or
8ample and 8ol'™t= to
5.0
Safety
ated in this
treated as a potential health
have been described (USNCI S8l
6'° Apparatus and Equipment
6.1. Apparatus —
however, each sample should be
for handling such materials
6.1.3 Bacterial filter holder—
6.1.4 Bacterial filters—
90
-------
Organic Residue Collection Unit
Regulators
Flow Pressure
Inlet
Glass Fiber ^
+/-3.0y Fluoropore K
0.45y Durapore JI
Silanized Glass Wool Column
40y Frits
XAD-2 Column
40y Frits
XAD-7 Column
Outlet
Figure 9. Schematic of nonvolatile residue organics concentration apparatus.
-------
VO
N)
Gauge 0-100 psi
Whitey
SS-IRM4-S4
Sample Chamber
Nylon
Male
Garden
Hose
1/4 in MPT
Veriflo
Model
IR401S250G
Exploded View
40 Micron Sintered
rStainless Frit —
1/4 in Swagelok
1 in Swagelok
Figure 10. Details of nonvolatile residue organics concentration apparatus.
(Note: prefiltration units are not shown.)
-------
6.1.4.1 Microfliter glass discs without binder resin (Millipore Type AP40,
AP40 047 05) for low total organic carbon filter holder.
6.1.4.2 Hydrophilic 0.45-micron Durapore filter (Millipore NVLP D4700 or
equivalent) for low total organic carbon filter holder.
6.1.4.3 Hydrophilic 0.45-micron Durapore filter (Millipore 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
Swagelok stainless steel fittings (Swagelok 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 Resin and glass wool columns—
6.1.6.1 Large sample volume (>50 L) columns of 200-cc bed volumes are
constructed, as shown in Figure 10, of 1-in by 26.5-in 316 stainless steel
tubing fitted with 1-in female Swagelok stainless steel fittings (Swagelok
SS1012-1-316, SS1613-1-316, SS1614-1-316 or equivalent) and a 1-in male
Swagelok stainless steel cap (Swagelok SS1610-C-316 or equivalent) modified as
follows. The center of the 1-in cap is tapped and threaded to accept a 1/4-in
pipe male coupling. The cap is fitted with a 1/4-in stainless steel pipe to
tube male coupling (Swagelok SS400-1-4-316 or equivalent) which is silver-
soldered in place. Prior to installation, the 1/4-in coupling is machined
internally from the tubing side for an opening 3/16-in wide and 3/4-in deep.
This opening is then fitted with a 5/32-in diameter by 1/8-in 40-micron
sintered 316 stainless steel frit. Both column ends are fitted with the
40-micron 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 shown in Figure 2 and described above, except that the columns are
constructed of 1/2-in by 13-in 316 stainless steel tubing fitted with 1/2-in
female Swagelok stainless steel fittings (Swagelok SS812-1-316, SS813-1-316,
SS814-1-316 or equivalent), the 40-micron sintered 316 stainless steel frits
and 1/2-in male Swagelok stainless steel caps (Swagelok SS810-C-316) modified
as described above. This entire column system is available from Tristate
Controls, Inc., 4303 Kellogg Avenue, Cincinnati, OH 45226.
6.2 Miscellaneous Apparatus Components—
(when line pressure of drinking water exceeds supplies 40 psi).
6.2.1 Pumping—The low line pressure drinking water sample source may be
pumped through the columns by connecting a control volume TFE diaphragm pump
(Milton Roy Co., model NR-117S or equivalent) between the drinking water
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 with a flow control valve, described in Section
93
-------
ls °sually
press
RS20 8tainleSs tel resrvoir or
f th
with
volUMS«60 1)
3
"«.d
• 2^3778,
EVaP"at0r <°W<»«ion Associate^ Inc.) N-Evaps Model 111 or
6.6 Analytical Balance - Readable to 0.01 mg »lth a pr.cl.ion of ± 0.01 m.
6.8 Special Glassware—
6.8.2 Soviet extraction apparatus. Corning series 3840 or equivalent.
°f Sustal"i"8 5<>» <=, for ... in glasstta«
(Amlcon
6.12 Cleaning of Apparatus and Glassware—
fol!,r WaSh* rinse with taP a°d ASTM Type I water
followed by successive rinses with pesticide-grade acetone and hexlne
94
-------
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 FR 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 XAD 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—
Silanized (Supelco No. 2-0411 or equivalent)
95
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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—
Prewashed 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
A?™3i V Particles; repeat the process with a second aliquot of
T TIT Vf*"'^ ^ 3 25"CC sta±nless steel c°!^. 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 the inlet end of the column •
to a 4-L stainless steel reservoir (Amicon Corporation, model RS4 or equiva-
lent). Successively wash the column with one liter each of pesticide-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-in stainless steel
Swagelok plugs for storage of the column until use (within one week).
8'° Residue Organics 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-in 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 prefiltration 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: the stainless steel frits
are omitted from the inlet end of the glass wool column. Label the 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, set the regulator to 30 to 35 psi. For use of the
large columns, adjust the flow rate to no greater than 250 mL/min by setting
the pressure regulator to no greater than 30 psi (Tabor and Loper 1984, Loper
et al. 1984). For use of the small columns, adjust the flow rate to no
greater than 100 mL/min.
96
-------
8 3 Apparatus for Drinking Water Sources at Line Pressures <40 psi—
The apparatus is assembled with proper flow directions for the
columns, as shown 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 psi. 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 may 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 8.2) to displace
the acetone.
8'4 SheTthf flow'rate'decreases to approximately 50% of the initial rate,
the bacterial filters probably need to be replaced. If the majority of the
sample, >90%, has been concentrated, continue collection. If not, then
discontinue the concentration operation, disassemble the bf'"^/^"^
holder and replace the filter(s) with fresh ones. The used f"£" «J Jlaced
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.
86 At the completion of a collection, the apparatus is disassembled in
the 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 the 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 et al. (1983, 1984) and
Tabor and Loper (1984).
97
-------
s*
eight
.
: =
1; s •
usins a r«Mn nf f J -, accomplished by Soxhlet extraction for 10 h
8.8 Concentration of Reduced Volume Extracts--
and rec0rded°Wi?hervoluLeVfPOr\tl0n' th* V°lume °f 6ach extract is B«e
(a) Registered trademark.
98
-------
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. At the time of bioassay, an aliquot of the residue solution is
removed from the sample vial; typically, this aliquot is adjusted to necessary
bioassay volume with DMSO. If it is necessary to know the mass of residue
organics per dose, the amount of organics should be determined gravimetrically
on a separate aliquot of the acetone solution of residue organics.
9.0 Quality Control
9.1 Solvent Blanks—
Samples of each lot of the hexane and acetone elution solvents and the
methylene chloride extraction solvent are concentrated for rautagenesis test-
ing. Two 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 mL via the micro-Snyder evaporative concentrator. The
residue concentrate is mixed with 400 pL of DMSO and submitted for muta-
genicity testing, two doses in duplicate, with 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 bioassay 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 organics is followed to determine contamination of the
cleaned XAD 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-mL 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 organics analysis procedure
(USEPA 1978). In this procedure, 5 yL of the extract are injected into a
flame ionization gas chromatographic unit fitted with a 6-ft by 4-mm glass
column containing 10% 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 10% full-scale eluting 5 min or later after injection.
The small sample volume apparatus (Section 6.1.6.2) is assembled as
described. Two 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 ±10%. Pass 1 L acetone through the columns,
followed by 40 L ASTM Type I water. Elute columns per Section 8.7, and
bioassay 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
99
-------
waters be dechlorinated with ferrous citrate fCheh et al
1979) 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 contain-In^ an inay* »*», /-~-n-_ . _,. ,.
-
-SPC un^r V,
-------
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.
ACS 1984 American Chemical Society. Division Environmental Chemistry.
Extended abstracts of papers - 188th National meeting Vol. 24(2), Washington,
DC: American Chemical Society.
Aiken GR, Thurman EM, Malcom RL, Walton HF. 1979. Comparison of XAD macro-
porous resins for the concentration of fulvic acid from aqueous solution.
Anal. Chem. 51(11):1799-1803.
Baker RA. 1970. Trace organic contaminant concentration by freezing. IV.
Ionic effects. Water Res. 4(8):559-573.
Baker RA, Malo BA. 1967a. Characterization of the microorganic constituents
of water by instrumental procedures. Purdue Univ. Eng. Bull. Ext. Ser.
129:252-262.
Baker RA, Malo BA. 1967b. Characterization of the microorganic constituents
of water by instrumental procedures. Water Sewage Works 114:223-230.
Baird R, Gute J, Jacks C. et al. 1980. Health effects of water reuse: A
combination of toxicological and chemical methods for assessment In
Jolley RL. Brungs WA, Gumming RB, Jacobs VA, eds. /ater chlorination
Environmental impact and health effects, Vol. 3. Ann Arbor, MI: Ann Arbor
Science Publishers Inc., pp. 925-935.
Baird RB, Jacks CA, Jenkins RL. 1981. A high-performance macroporous resin
concentration system for trace organic residues in water In: Cooper WJ, ed.
Chemistry in water reuse, Vol. 2. Ann Arbor, MI: Ann Arbor Science
Publishers, Inc., pp. 149-169.
Baum RG, Saetre R, Cantwell FF. 1980. Liquid chromatography columns of
microparticle Amberlite XAD-2. Anal. Chem. 52(1):14-19.
Braus H, Middleton FM, Walton G. 1951. Organic chemical compounds in raw
and filtered surface waters. Anal. Chem. 23(8): 1160-1164.
Buelow RW, Carswell JK, Symons JM. 1973. An improved method for determining
organics by activated carbon absorption and solvent extraction. J. Am. Water
Works Assoc. 65:57-72.
101
-------
Burnham AK, Calder GV, Fritz JS, et al. 1972. Identification and estima-
tion of neutral organic contaminants in potable water. Anal. Chem.
44(1):139-142.
Carbridenc R, Sidka A. 1979. Extraction of organic micropollutants from
water for the purpose of performing biological assays. Presented at the
European Symposium on the Analysis of Organic Micropollutants in Water,
Berlin, Federal Republic of Germany. (In French, translation).
Care R, Morrison JD, Smith FJ. 1982. On limits of detection of traces of
volatile organics in water, using Amberlites XAD-2 resin. Water Res.
16:663-665.
\
Cheh AM, Skochdopole J, Koski P. 1979. Nonvolatile mutagens in drinking
water: production by chlorination and destruction by sulfite. Science
207:90-92.
Chriswell CD, Ericson RL, Junk GA et al. 1977. Comparison of macroreticular
resin and activated carbon as sorbents. J. Am. Water Works Assoc
69:669-674.
Coleman WE, Melton RG, Slater RW et al. 1981. Determination of organic con-
taminants by the Grob closed-loop-stripping technique. J. Am. Water Works
Assoc. 73:119-125.
Council on Environmental Quality. 1979. Environmental Quality, the 10th
Annual Report of the Council on Environmental Quality. Washington, DC: US
Government Printing Office.
Council on Environmental Quality. 1980. Environmental Quality, the llth
Annual Report of the Council on Environmental Quality. Washington, DC: US
Government Printing Office.
Council on Environmental Quality. 1981. Environmental Quality, the 12th
Annual Report of the Council on Environmental Quality. Washington, DC: US
Government Printing Office.
Council on Environmental Quality. 1982. Environmental Quality, the 13th
Annual Report of the Council on Environmental Quality. Washington, DC: US
Government Printing Office.
Crathome B, Watts CD, Fielding M. 1979. Analysis of non-volatile organic
compounds in water by high-performance liquid chromatography. J. Chromatoer.
185:671-690.
Crump KS, Guess HA. Science Research Systems, Inc. 1980. Drinking water
and cancer: Review of recent findings and assessment of risks. Prepared for
Council on Environmental Quality - Springfield, VA: National Technical
Information Service, US Department of Commerce, p. xiii. Contract No.
EQ10C018.
102
-------
A note on the losses of monosaccharldes, amino
« ««t. during concentration procedures. Anal.
Biochem. 84:186-190.
Deinzer M, Melton R, Mitchell D. 1975. Trace organic ^aininants in
drinking water; their concentration by reverse osmosis. Water Res.
9 (9): 799-805.
Dressier M 1979. Extraction of trace amounts of organic compounds from
water with' porous organic polymers. J. Chromatogr. 165:167-206.
Fritz JS. 1976. Concentration of solutes from aqueous solution. Accounts
of Chem. Res. 10:67-72.
Garrison AW. 1977. Analysis of organic compounds in water to support health
effects studies. Ann. NY Acad. Sci. 298:2-19.
Glatz BA, Chriswell CD, Arguello MD. 1978. ^™*±™o*?
for mutagenic activity/ J. Am. Water Works Assoc. 70:465-468.
Grieser MD, Pietrzyk DJ. 1973. Liquid ch romatography on a porous
polystyrene - divinyl benzene support. Anal. Chem. 45(8) : 1348-1353.
Hindin E, Bennett PJ. Narayanan SS. 1969 -Organic compounds removed by
reverse osmosis. Water Sewage Works 116:466-470.
Kites RA 1977. The analysis of organic water pollutants by gas chroma-
tography and gas chromatography - mass spectrometry. Advances in
Chromatograph 17:69-129.
Hoffman GR. 1982. Mutagenicity testing in environmental toxicology.
Environ. Sci. Technol. 16(10) :560A-572A.
James HA Steel CP, Wilson I. 1981. Impurities arising from the use of
Sl-2 resin for the extraction of organic pollutants in drinking water. J.
Chromatogr. 208:89-95.
Jenkins RL, Jacks CA, Baird RB et si. 1983. Mutagenicity ^organic solute
recovery from water with a high-volume resin concentration. Water Res.
17(11):1569-1574.
Johnston JB, Herron JN. 1979. A routine water monitoring test for mutagenic
compounds. UIUC-WRC-79-0141. University of Illinois. Urbana, IL.
Johnson JB, Verdeyen MR. 1981. Recovery of mutagens from water by the
Sit/distillation method. In: Cooper WJ, ed. Chemistry in water reuse,
Vol. 2. Ann Arbor, MI: Ann Arbor Science Publishers, Inc., pp. 171-iyu.
Jolley RL, Katz S, Mrochek JE. 1975. Analyzing organics in complex, dilute
aqueous solutions. Chem. Technol. 5(5) : 312-318.
103
-------
ln water for blol°8icai
nalvs ff » • 1974' Use °f -"croretlcular resins in the
analysis of water for trace organic contaminants. J. Chromatogr. 99^745-762.
I'8'' Advm"s ln the identification and analysis of organic
" Vo1- l and 2- Ann Arbor- MI: ic
for'thf'dlr ?JelJ1fCPf ^ Kranen HJ 6t al- 1981a' The use of XAD-res
for the detection of mutagenic activity in water. Chemosphere 10:85-98.
^
Kool HJ, Van Kreijl CF, Van Oers H. 1984. Mutagenic activity in drinkine
1 ** * SUrVS ^ 3 "r" *
Kopfler FC, Coleman WE, Melton EG et al. 1977. Extraction and iden-
icad?1ci? 298"S? »I"^°llu"nt- "-*"« "—sis method. Ann. NY
Kopfler FC 1980. Alternative strategies and methods for concentratln.
chemicals from water. In: Waters MD, Sandhu SS, Huelsinjjh JL Cllxto"
'll N.wh?orfer?,B10aSr^/,n the AnalySlS °f C0°"1
ll. New York: Plenum Publishing Corp. , pp. 141-153.
'.han? °?i' KyrlaZ^-A- 1980' Tum°^ transformation and latency
r l P 7 "ansfomed and ^rphologically normal BALB/3T3 cells in
m Meetlng T±SSUe CultUre Asso^ation, June, St.
ce , S 6t a1' 198°- Mali8"^t transformation of
Exp oxi"l l!^ or»toic- "**««• ««« drinking water. J. Pathol.
atinnf ™- 1979' I-olation and con-
T f or8an°PhosPhorus pesticides from drinking water at the ng/L
. J. Assoc. Off. Anal. Chem. 62(2) :241-249.
104
-------
Toxicol. Environ. Health 4:919-938.
Loper JC. 1980. Mutagenic effects of organic compounds in drinking water.
Mutat. Res. 76:241-268.
Inc. pp. 1199-1210
carbon treatment system. Environ. Sci. Technol. (i
Maciorowski AT. Little LW, Raynorl*. 19| 52 Bioassays-procedures and
results. J. Water Pollut. Contr. Fed. 54(6) :830-848.
Malcolm RL, Thurman EM, Aiken GR. 1977. The concentration and fractionation
'
314.
.
mental Health-XI. Columbia, MO: University o
McGuire MJ, Suffet IH. 1983. Treatment of water by granular activated car-
bon. Washington, DC: American Chemical Society.
M.ier JR Bishop DF. 1984. Effectiveness of. conventional treatment processes
for removal of mutlgenic activity from municipal wastewaters. Environ.
Mutagen. 6:451.
Middleton FM, Grant W, Rosen AA. 1956. Drinking water taste and odor. Ind.
and Eng. Chem. 48(2) : 268-274.
Middleton FM, Stephan DG. 1962. Water renovation. Chem. Eng. Progress
58(10) :63-68.
Mieure JP, Dietrich NW. 1973. Determination of trace organics in air and
water. J. Chromatogr. Sci. 11:559-570.
Modell M, deFilippi HP. Krukonis V. 1978. Regeneration of activated carbon
"ith supercritical carbon dioxide. Presented before the Division of
Environmental Chemistry. Miami, FL: American Chemical Society.
NAS-NRC. 1977. Drinking water and health. Vol. 1. Report on the safe
drinking water committee, Board of Toxicology and Environmental Health
Hazards! Istmbly of Life Sciences. Washington, DC: National Academy Press.
NAS-NRC. 1980a. Drinking water and health. Vol. 2. Report °n the s
drinking water committee, Board of Toxicology and Environmental Health
Hazards! Assembly of Life Sciences. Washington, DC: National Academy Press.
105
-------
y 1
Hazards, Assembly of Life Sciences, Washington, DC: tio^Llmy Press.
NAS-NRC. 1982a. Drinking water and health. Vol. 4. Report on the safe
drinking water committee, Board of Toxicology and Environmental Health
Hazards, Assembly of Life Sciences. Washington, DC: National AcademJ Press
NAS-NRC. 1982b. Quality criteria for water reuse. Panel on quality cri-
SisfLWo^feT<' ^ °* T°xlcol°^ and Environmental Health Hazards
Commission on Life Sciences, Washington, DC: National Academy Press.
NAS-NRC. 1983. Drinking water and health. Vol. 5. ' Report on the safe
Lards'8 EZ^^S' 1°?* °f Toxicol°Sy «* Environmental £ll h
Hazards, Assembly of Life Sciences, Washington, DC: National Academy Press.
Nellor MH, Baird RB, Smyth JR. 1984. County Sanitation Districts of Los
Angeles County. Health effects study. Final report. Whiter cl- Countv
Sanitation Districts of Los Angeles County. wnittier, CA. County
Nestmann ER, LeBel GL, Williams DT, Kowbel DJ. 1979. Mutgenicitv of
S
J> ^T* A; 1983< Isolation * organic compounds
2^17: "" tOX±COl°^Cal ^^' !«.». 5.
tiles for J01123162 LF' Ja^ J* 1984' ^-Concentration of headspace vola
chromatography. J. Chromaiogr.
w %* M h1977a' Am?erlites ^ copolymers in reversed phase
49(6):757-l4 Press«re liquid chromatography. Anal. Chem.
Pietrzyk DJ Chu C. 1977b. Separation of organic acids on Amberlites XAD
8^
Pietrzyk DJ, Kroeff EP, Rotch TD. 1978. Effect of solute ionization on
copolymers. ^
Keith ttd /97T^ T^/0ufations of organic pollutant analysis. In: L.H.
. Ann
o ' Sconce iT "** "^^ ^ c-««»tion of dilute
106
-------
Simmons VF, Tardiff RG. 1976. Mutagenic activity of drinking water con-
centrates. Mutat. Res. 38:389.
r 1978 Strategy for collection of drinking water concentrates. In
-
USEPA.
Environ. Sci. Technol. 10:1273-1275.
Tabor MW Loper JC. 1980. Separation of mutagens from drinking water using
coupLrbioassay/analytical fractionation. Int. J. Environ. Anal. Chem.
8:197-215.
Tabor MW. 1983. Structure elucidation of 3-(2-chloroethoxy)-l,2-Dichloro-
propene! a new promutagen from an old drinking water residue. Environ. Sex.
Technol. 17(6) : 324-328.
Tabor MW, Loper JC. 1984. Analytical isolation, separation, and iden-
tmcation of mutagens from nonvolatile organics of drinkmg water. Int. J.
Environ. Anal. Chem. (in press).
Thurman EM, Malcolm RL. Aiken GR. 1978. Prediction of capacity for aqueous
organic solutes absorbed on a porous acrylic resin. Anal. Chem.
50(6):775-779.
iLSiJrs-i^Jsss; XL ^^L=tst^r^L
University of Cincinnati. Chicago, IL: IIT Research Institute.
USEPA 1978 U. S. Environmental Protection Agency. Office of Research and
Development IERL-RTP Procedures Manual: level 1 environmental assessment
(2nded.). Washington, DC: USEPA-600/7-78-201. PB-293795.
USEPA. 1979. U. S. Environmental Protection Agency Office of Re -arch and
Development. EMSL-CIN. Chlorine, Total Residual, Method 330.2 ^^^Inta
Chemical Analysis of Water and Wastes. Cincinnati, Ohio: U. S. Environmenta
Protection Agency, pp. 1-5. USEPA-600/4-79-020.
USEPA 1983. U. S. Environmental Protection Agency. Office of Research and
Devetpment? Research Outlook, 1983. Washington, DC: ^'l'^"^
Protection Agency. USEPA-600/9-83-002. Accession No. PB-83 172510.
107
-------
Van Der Gaag MA, Noordsij A., Poels CLM, Schippers JC. 1982. Orienterend
onderzoek met analytisch-chemische en genotoxicologische meetmethoden naar
het effect rian waterbehandelingsprocessen. H,0 15:539-558. (in Dutch).
Van Rossum P, Webb RG. 1978. Isolation of organic water pollutants by XAD
resins and carbon. J. Chromatogr. 150:381-392.
Watts CD Crathores B, Fielding M et al. 1982. Nonvolatile organic compounds
in treated waters. Environ. Hlth. Perspect. 46:87-99.
Webb RG. 1975. U. S. Environmental Protection Agency. Southeast Environ-
mental Research Laboratory. Isolating organic water pollutants: XAD resins,
urethane foams, solvent extraction. Final report. Springfield, VA: NTIS,
U. S. Department of Commerce, EPA-660/4-75-003. Accession No. PB-245 647.
Williams DT, Nestmann ER, LeBel GL, Benoit FM, Otson R. 1982. Determination
of mutagenic potential and organic contaminants of Great Lakes drinkine
water. Chemosphere. 11(3):263-276.
Yamaski E, Ames BN. 1977. Concentration of mutagens from urine by adsorption
with the non-polar resin XAD-2: Cigarette smokers have mutagenic urine.
Proc. Natl. Acad. Sci. U.S.A. 74:3555-3559.
Zoeteman_BCJ Hrubec J, de Greef E, Kool HJ. 1982. Mutagenic activity asso-
ciated with by-products of drinking water disinfection by chlorine, chlorine
dioxide, ozone and UV-Irradiation. Environ. .Hlth. Perspec. A6-197-205
108
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SECTION 4
ENVIRONMENTAL WATERS AND WASTEWATER
REVIEW OF THE LITERATURE
Introduction
Genotoxic agents are known to exist at very low concentrations in waste-
water (Saxena and Schwartz 1979). However, mutagens, even potent ones,
present at ppm and ppb levels may not be detectable when water samples are
tested directly in sensitive bioassays such as the Ames test, since they fall
below the sensitivity level for the bioassay. Consequently, special process-
ing techniques are used to concentrate dilute wastewater samples before they
are evaluated in such biological screens.
The rationale behind concentrating samples prior to subjecting them to
bioassay procedures is that organisms in the aquatic food chain are known to
bioconcen?rate chemicals in a manner that might effectively yield hazardous
levels of genotoxic agents when those organisms or animals which feed upon
them are consumed by humans. A second reason is that long-term exposure of
humans to very small quantities of water contaminants might result in high
body burden if the chemicals are stored.
A primary objective in wastewater preparation and testing for biological
activity is to collect samples that are representative of the waste effluent
and to recover the bioactive molecules contained in the water in a state which
retains their concentrations relative to each other. It is also important to
prevent the formation of new combinations that might produce activity not
initially present in the dilute wastewater.
Two approaches have been described for the processing of water samples
for bioassay. The first involves concentration of all solubilized or sus-
pended materials by removing only the water, and the second selectively
removes the organic substances from the water. Examples of these two
approaches, with their respective advantages and limitations are listed in
Table 1 It is possible to combine methods which may improve the recovery ot
organic molecules from the water. Combination methods are useful when very
large quantities of material (>50-L samples) need to be processed. Kopfler
(1980) has described four methods which combine concentration and organic
compound removal by ion exchange and/or XAD resins.
109
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TABLE 1. METHODS PROPOSED FOR CONCENTRATION AND ISOLATION
OF CHEMICALS IN DILUTE WATER SAMPLES
Method
Advantages
Limitations
Freeze concentration - a vater
•ample la frozen Into a shell
of pure Ice with the dissolved
substances In the center un-
frozen area.
Freeze-drylng - removal of
vater In vapor formed by freez-
ing under vacuum. The solids
suspended or solublllzed In the
water are left behind.
Reverse osmosis - water is re-
moved by applied pressure
•cross a membrane. The
physical properties of the mem-
brane determine the molecular
weights of the materials remain-
Ing after the water is removed.
Direct liquid-liquid extraction
- organics can be Isolated from
water by comlxing immiscible
organic solvents with
wastewater. The organics will
become solubilized In the
organic solvent vhlch can be
easily separated from the
mixture.
Isolation of organics on
activated charcoal - water is
passed over activated charcoal.
The charcoal is then processed
by Soxhlet extraction In
organic solvents to recover
organics.
Permits recovery of all dis- Recovery of organic solutes
solved materials Including vola- decreases with increaalng salt
tiles and ionized species. content of the samples due to
alteration of ice formation.
The level of concentration may
be too low for recovery of very
dilute species.
Recovery
Evidence for up to 80* recovery
of organics materials in a
reconstruction experiment.
Inorganic species will also be
present in the concentrate.
Recovers all species including
organic and inorganic chemi-
cals. Equipment Is available
to freeze dry large volumes
(20-40 L) of collected water.
The level of concentration Is
very high by the removal of all
water.
Commercial systems capable of
handling large volumes are
available. A large portion of
ionic species and organic car-
bon species are retained In the
concentrate.
The process is direct and can
be used with relatively large
volumes (several liters).
Reflexlng of the solvent can
concentrate the Isolated
organics Into even smaller
volumes.
The process perferentlally
concentrates organics on a
solvent phase.
Chemicals which are volatile at
temperatures and pressures used
will be lost. Since all dis-
solved chemicals will be re-
covered it is very difficult to
remove the organics from Inor-
ganics. The process is time
consuming.
The process can be time consum-
ing. Since both Inorganic and
organic compounds are concen-
trated, a second process to
separate these two classes
would be required. Some com-
mercial systems are not
amenable to chlorinated water
samples.
Extraction of large volumes
requires large amounts of
solvent. The level of
extraction Is moderate since
the process would require
continuous removal and addition
of fresh solvent to the water
sample until all organics have
been Isolated. Concentration
or organics by refluxing the
solvent samples requires
special equipment and may
result in undesired chemical
reactions during the heating
process.
Carbon recovery is not as good Not reported.
as other solid phase media.
The requirement for air drying
and Soxhlet extraction with
organic solvents might lead to
artifact production.
Recovery of 60Z-80Z of dis-
solved material is possible de
pending on the type membrane
selected.
Not reported.
Method of Reference
Shapiro. J. 1961.
Freezing out, a safe
technique for con-
centration of dilute
solutions. Science
133:2063-2064.
Virtually 100Z of all dissolved
materials which are not vola-
tile.
Kopfler et al. 1977.
Extraction and iden-
tification of organic
mlcropollutants: re-
verse osmosis method.
Ann. N.T. Acad. Scl.,
298:20-30.
Buelow et al. 1973.
An improved method
for determining
organics by activated
carbon adsorption and
solvent extraction.
J. Am. Water Works
Assoc., 65:57-72.
continued-
-------
TABLE 1 continued-
Sorbant resin Isolation of
organlcs - this method utilizes
a vide range of porous resins
to recover organic compounds
from water samples.
The resins commonly used (i.e.,
XAD-2 have great affinity for
hydrophoblc substances and
retain virtually all of organic
molecules. Once the water
sample has passed over the
resins, the trapped organlcs
can be eluted from the resin.
This process does not require
Soxhlet extraction at high
temperatures. The process can
easily handle very large water
samples and Is relatively
Inexpensive.
Although resins are effective
in adsorbing organic molecules,
it is possible that not all
organics of biological interest
are adsorbed. If they are, it
is possible that they cannot be
eluted from the resin by the
solvents used. Resin selection
and preparation of columns for
concentration.
50* to 90Z of organics present
in a sample can be recovered
depending upon the parameters
employed.
Junk, et al. 1974.
Use of macroreticular
resins in the analy-
sis of water for
trace organic con-
taminants. J. Chro-
matogr.. 99:745-762.
-------
Objectives
The objective of this section is to review the methods for environmental
and wastewater (referred to as wastewater) processing and recommend a
method that will provide a convenient and reliable procedure to concentrate
genotoxic chemicals suitable for Ames test evaluation.
al
methodology should meet a set of criteria proposed by Ross et
1. Concentrate relatively large samples.
2. Provide for a concentration factor >200 times.
3. Preserve volatile compounds. =
4. Preferentially concentrate and extract potential genotoxic agents
5. Reduce artifact formation.
6. Maintain relative concentration of all compounds.
7. Employ reagents that are compatible with bioassays.
8. Produce a relatively sterile test sample.
It is unlikely that any single method, or even combinations of methods,
described in Table 1 would be able to meet all eight of these criteria-
however, it should be possible to describe a method which achieves most of
the desired properties.
Previous Studies
There are published reports describing water processing for various
purposes, including the preparation of samples for bioassay (EPA 1977, Junk et
toon! T R°!S et a1' 198°' L°Per et al- 1978» Rappaport et al. 1979, Kopfler
IVBU;. in virtually all of these methods, sorbent columns were used to
concentrate the organic compounds which are the most likely candidates for
genotoxicity. At least one of the studies (Ross et al. 1980) conducted tests
for recovery of organic mutagens from spiked water samples as a means to
estimate the reliability of the proposed techniques. In other studies, the
methods were applied to actual wastewater samples collected from rivers and
lakes. For example, pulp and paper mill effluents contain materials which are
mutagenic in the Ames test (Nestman et al. 1979). The investigators in this
study identified at least one mutagen, the resin neoabietic acid, in the
effluents from a paper recycling plant. Other mutagens were obtained from
solid waste collected from effluents emanating from a paper recycling plant
(Klekowski and Levin 1979). In these studies, the mutagens could be collected
as solids suspended in the water and did not require elaborate sample
handling. v
Follow-up studies to the wood pulp and paper making reports showed that
chlorination of organic compounds derived from lignin stabilized their muta-
genic activity (Nazar and Rapson 1980). Further studies along this line have
shown that substitution of chlorine dioxide for aqueous chlorine or elevation
of pH by adding lime to the treatment plant will substantially reduce the
mutagenicity of wastewater (Nazar and Rapson 1980, Eriksson et al 1979) in
laboratory studies, Nazar and Napson (1982) showed that several Ames test
mutagens produced during the bleaching of wood pulp (i.e., dichloroacetone,
112
-------
tetrachloroacetone and o-benzoquinone) could be inactivated by raising the pH
of the treatment solution to pH 10 with NaOH. The mechanism appears to be by
cleavage of organically bound chlorine by hydroxide ions.
In another study employing concentrations of 200-L samples of drinking
water river water and well water on XAD-2 resin columns, Nestmann et al.
(1979) > *ere able t0 <^°«s«ate the P"8611" °f bac?er1^ mUJag?n! HSfrom
strains TA-98 and TA-100. Bacterial mutagens have also been isolated from
swimming pool water by Honer et al. (1980).
In a more recent study conducted on water samples collected from a lake
in Illinois, the investigators identified mutagens active in strains TA-98 and
TLJoJ Sartlein et al. 1981). The investigators in this study concentrated
the water 3,000-fold on XAD-2 resin and performed preincubation tests with the
eluents The peak recovery of mutagens coincided with the application of
^-emergent herbicides used on nearby farms and probably involved Caching of
herbicidJe or breakdown products from the fields into tributarie * ^^ ^
lake. Gas chromatography of selected lake water samples showed the presence
of several pesticides, including some which are known to be mutagenic in
Salmonella.
Chemical analysis of wastewater from trinitrotoluene (TNT) production
suggests the presence of a large number of potential "i"03™^!™^^.
WhS the wastewater components were combined into a syn*hef
-------
TABLE
2. ORIGIN OF INDUSTRIAL WASTEWATER EFFLUENTS TESTED IN
THE AMES ASSAY
Sample
Number
Industry Type
Raw Material
Product Description
Source of
Water
100
101
102
103
104
105
106
107
108
109
110
Pulp and Paper
Plastics,
Synthetic Fibers
Textile
Petroleum
Refining
Textile
Paper Boar Mill
Pulp and Paper
Synthetic Resins
Tanning
Chemical
Manufacturing
Unknown
Wood fibers
Velveteen, corduroy,
cotton, dyes, pigments
Venezuelan and
Mideast crude
Cotton, polyester, wool
1000 TPD hardwood chips,
NaOH, Na
1200 TPD virgin material
130 TPD recycled hard-
wood, pine and corrugated
paper
131,000 cattle hides
Paper products, bleached wood
Finishing and dying of textiles
Gasoline and heating oil
Dying, finishing, printing
fabrics
Manufacture of corregated medium
from pulp produced from non-
sulfur, semi-chemical pulping
process
Unbleached craft paper corrugated
medium, bleached market pulp
Synthetic resins and fibers
Vegetable and mineral tanning of
brine hides; curling finishing
hides - leather converters
Manufacturing industrial
inorganics and amines
River
Deep wells
River
City supply
and river
water
River
River
Wells
Wells and
springs
Wells, creek
River
Unknown
(a) Each sample consisted of two gallons of wastewater.
-------
TABLE 3 CORRELATION OF GENETIC ACTIVITY IN SALMONELLA ASSAY
TO EXTRACTABLE ORGANICS FROM CHESAPEAKE BAY
SAMPLES AND SPIKED SAMPLES
Sample
SPike(f)
100
101
102
103
104
105
106
107
108
109
110
mg OrganicsA
mL Extract
0.063
1.600
7.100
0.500
1.900
0.200
1.000
1.300
<0.100
0.100
<0.100
0.900
MEG (yg,ger
Plate) I
1.575
320.000
710.000
12.500
95.000
20.000
50.000
130.000
20.000
> 20. 000
>20.000
>180.000
Revertants,
per Plate^
Strain
(e)
Specific, v
Activities
Revertants
per mg Organics
510.0
241.0
257.0
198.0
213.0
42.5
72.5
34.5
31.5
18.0
6.0
17.0
TA-100
TA-100
TA-100
TA-100
TA-100
TA-98
TA-98
TA-98
TA-98
TA-98
TA-98
TA-98
323809.5
753.1
361.9
15840.0
2242.1
2125.0
1450.0
265.4
1575.0
1900.0
300.0
94.4
(a) Revertants per plate (d) x 1,000 divided by MEG (c).
(b) Total organics extracted from one gallon of the sample. Final extract
(c) MEC^nimtm effective concentration which exhibited a minimum of two-
fold increase in the number of revertants over the background.
(d) Revertants per plate in the presence of S-9 minus the spontaneous
(e) Strain*?™; which the data was taken to extrapolate the specific activity.
(f) A one-gallon sample was spiked with 492 yg of N-nitroquinoline-N-oxxde
to serve as a known source of mutagens.
115
-------
TABLE 4. COMPARISON OF AVAILABLE PROCESSING METHODS
Criteria
Freeze Freeze-
Concentration Drying
Direct
Liquid- Activated Sorbant
Reverse Liquid Charcoal Resin
Osmosis Extraction Isolation Isolation
Concentrate large samples
Provide >200 times
concentration
Preserve volatiles
Preferentially concentrate
genotoxicants
Reduce artifacts
Maintain relative concentra-
tions of chemicals
Employ biocompatible reagents
Produce a sterile test sample
(a)
(a)
(c)
(a)
(d)
(c)
(d)
(b)
(b)
(d)
(a)
(a)
(c)
(a)
(d)
(a)
(c)
(c)
(c)
(b)
(c)
(e)
(d)
(b)
(a)
(b)
(b)
(c)
(c)
(e)
(c)
(c)
(b)
(b)
(b)
(c)
(c)
(e)
" (c)
(c)
(c)
(d)
(b)
(d)
Cc)
(e)
(c)
(c)
(a) Not a useful method.
(b) Poor method.
(c) Good method.
(d) Excellent method.
(e) Unknown.
-------
Conclusions
inexpensive methods employing sorbent resins are readily available
personal communication)
wastewafer analysis and is recommended as the method of choice
117
-------
REFERENCES
Brusick DJ, Young RR. 1981. Level 1 bioassay sensitivity. EPA-600/7-81135
7
the
Heartlein MW, DeMarini DM, Katz AJ, Means JC, Plewa MJ
Junk GA. Elchard JJ, Grleser MD, Wltiak D, wttlak Jl.
MD
Klekowski E, Levin DE. 1979. Mutagens
recycling wastes: Results of
Mutagenesis 1:209-219.
Kopfler FC 1980. Alternative strategies and methods for concentrating
chemicals from wat-Pi-. In: sh—*--'r *>J • • ^-""l-eui-rati"g
II. V _. _f __iiu af
>, eds. Plenum Press, New York. Environ. Sci. Res!
118
-------
Nazar MA, Rapson WH. 1982. pH stability of some mutagens P"duced by
aqueous chlorination of organic compounds. Environ. Muta. 4:435-444.
Nestmann ER, LeBel GL. Williams DT, Kowbel DJ. 1979. Mutagenicity of organic
extracts from Canadian drinking water in the Salmonella/ mammalian-microsome
assay. Environ. Muta. 1:337-345.
Nestmann KR. Lee EGH, Mueller JC, Douglas GR. 1979. Mutagenicity of resin
allls identified in pulp and paper mill effluents using the Salmonella/
mammalian-microsome assay. Environ. Muta. 1:361-369.
Rappaport SM, Richard MG, Hollstein MC, Talcott RE. 1979 Mutagenic activity
in organic wastewater concentrates. Environ. Sci. Technol. 13:957-961.
Rapson WH, Nazar MA, Butsky W. 1980. Mutagenicity produced by Aqueous
chlorination of organic compounds. Bull. Environ. Contam. Toxicol. 24:590.
Ross WD. Hillan EJ, Wininger MY, Gridley K, Lee LF, Hare RJ, Sandhu SS 1980.
Aqueous effluent concentration for application to biotest ^sterns In.
Short-Term Bioassays in the Analysis of Complex Environmental Mixtures II.
Vol. 22. Waters MD, Sandhu S, Huisingh JL, Claxton L, Nesnow S, eds. Plenum
Press, New York. Environ. Res. pp. 189-199.
Saxena J, Schwartz DJ. 1979. Mutagens in wastewaters renovated by advanced
wastewater treatment. Bull. Environ. Contam. Toxicol. 22:319-326.
Spanggord RJ, Mortelmans KE, Griffin AF, Simmon VF 1982. Matagenicity in
Salmonella typhimurium and structure-activity relatxonshxps of ^stewater
components emanating from the manufacture of trinitrotoluene. Environ.
Muta. 4:163-179.
119
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ENVIRONMENTAL WATERS AND WASTEWATER WORKGROUP REPORT
Workgroup Tasks
Definitions of Wastewater and Wastewater Components-
solids^ JssiSnS^r^sn::; y% 2°: ^lnking water to
that can be processed. divided into zones and components
the method °f
of thebul ar met° or ""-T separation
the
The sixth task was to propose standard quality control procedures for:
1. Sampling
2. Phase separation
3. Extraction and concentration
4. Bioassay
Peer Review Comments —
All peer review comments were incorporated into the final protocol.
Workgroup Progress —
a. Definitions
120
-------
. Suspended nonaqueous phase - suspended nonaqueous liquids.
b Figure 11 defines how the collected sample will be separated into
three phases and how these phases will be processed prior to
bioassay.
Methods identified by footnotes are written elsewhere in this
report .
Summary of Key Workgroup Discussions
C°nSeS«ste»«ei°in »ltlptu.le medium which 11*. naturally to other
protocols.
Unresolved Issues —
a. Spiking of samples
1 . Examples
2. When to add
b. Quantify mutagenic activity
1. Revertants/mg total organics recovered from aqueous phase.
2. Revertants/mg total organics recovered from nonaqueous liquid
phase.
Revertants/mg total organics recovered from suspended solid.
Revertants/mg total organics recovered from sedimental solids.
Revertants/mg total organics recovered from all sources.
6. Revertants /liter of sample (unprocessed).
7. Revertants/liter/hour of unprocessed emission.
c. Blanks - types, use, source, QC
d. TOC on initial sample?
e. Safety of methylene chloride washing
Evaluation of Proposed Protocol
A draft protocol was prepared and reviewed by members of the workgroup.
Other Data or Information Requirements
3.
4.
5.
Information Needs—
a. Effects of matrix on mutagenic activity.
121
-------
Nonaqueous Liquid
Phase
I
Collected Sample
24-h Gravity Separation
Aqueous Liquid
Phase
Weigh and Determine
Volume
Solid Sediment
Phase
Weigh and Determine
Volume and Percent
Suspended Solids
/ N
(a), <5* Solld* >5% Solids
Wel8ht by Weight
Weight Determination
Process by Nonaqueous
Liquid Wastes Protocol
(b) Liquid-Liquid
Extraction
Process by
Drinking
Water
Protocol
(c) Filter
or Centri-
fuge Sample
to Separate
Liquid from
Suspended
Solids
Process by
Waste Solids
Protocol
Bioassay
Bioassay
Concentrate
Solvent
Exchange
I
Bioassay
/ \
Bioassay
Liquid Solids
I
Process
by Drinking
Water
Protocol
I
Bioassay
Process
by Waste
Solids
Protocol
\
Bioassay
Note:
bioassay
- "egat^vf (not ""tagenlc) it is recommended that
Sub3e«ed to (a) or (c) processing and retested in the
Figure 11. Wastewater sample processing flow diagram.
122
-------
b. Methods for bioactivity expression and what it means.
methods for sample collection, transport and storage - EPA
group should be formed to do this.
b. How much chemistry data on samples should be developed?
123
-------
WASTEWATER FOK
1.0 Scope and Application
2-0 Summary of Method
3.0 Definitions
nonaqueous
and
in addition to the aqueos and souphases
" A ?"", C°mp°Und added to a *™P** i« ^own amounts
C°ncentration -asurements of other compounds that are
the Same tlme'
under
Preservation and storage, as vel as with
Reagent Blank - Reagent water placed in a sample container in the
siteV^M" " ' Sample ±n &U resP-ts,'including exposure to
site conditions, storage, preservation and all analytical procedures.
124
-------
Nonaqueous Liquids Phase - Liquids whose major component is not water
and which form discrete zones in an aqueous medium.
Sediment Solid Phase - The solid or semisolid phase recovered from a
24-h resting wastewater sample.
4.0 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 by 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 reagent blanks.
4 4 Matrix interferences may be caused by contaminants that are contracted
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 should
be employed to evaluate matrix interference.
4 5 Methylene chloride can interfere with the mutagenicity assay, so care
must be exercised to completely remove all methylene chloride prior to
dilution with dimethyl sulfoxide.
4 6 The sample will be supplied for Ames testing in its most concentrated
form in dimethyl sulfoxide, because additional sample concentration in the
presence of dimethyl sulfoxide (B.P. 189 C) is prohibitive due to P°t«tial
thermal or evaporative loss of sample organic constituents. If the extract
not soluble in its concentrated form, dilution with additional dimethyl
sulfoxide or other appropriate solvents is advised.
4 7 Some extracted concentrated samples in DMSO 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 alternative
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.
is
125
-------
5.0 Safety
reduce'j to the
-a
to safety procedures consistent with this fact (NCI 1981).
5.5 Care should be used when pressurizing or vacuum-filtering
fxouLlr TSSUre °r V3CUUm mlght CaUSe Vessel or filter breaka
expulsion of concentrated sample into the laboratory environment;
desticated'ani/r'^3'" ^^^ ™7 C™e fr°m SOUrCes confining human or
domesticated animal excrement, communicable disease risk is present Labor*
tory personnel should be adequately immunized for communicable diseases? and
all^samples should be considered potentially contaminated and handled accord-
6.0 Apparatus and Equipment
6.1 Grab Sample Bottle —
6.2 Pressure Filter —
Millipore unit fitted with a glass fiber filter (1-p pore size).
6.3 Centrifuge —
fue'' ' refrigerated SaffiPle Centrifuge capable of handling 500-ml
6.4 Celite column to trap partlculate matter prior to resin concentration
concentration
centrifuge tubes.
6.4 Celite colu
. - - — rr-—"-"-"— •—*•*•*•«"**•*•• "*«* t. u d- u
(acceptable devices are commercially available).
126
-------
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-mm 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.
6.5.4 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. (Hershberg-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
(± 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.
127
-------
7.3 Sodium Thiosulf ate (ACS), granular.
7.4 Sulfuric Acid Solution (1+1) —
Slowly add 50 ml of H2S04 (sp. gr. 1.84) to 50 mL of reagent water.
Sulfoxide (Pesticide-
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—
other aL™-"-0*"* """^ " I0° U8/L °f »«»" «*»• «
8-° Quality Control
aM11\Xf-vBJf0re performing any analyses, the analyst must demonstrate the
ability to generate acceptable accuracy and precision with this method?
r^c°8nltion of the rapid advances that are occurring in chroma-
^••T—.^^^ ^sssi- ~
method, the analyst ls required to repeat the procedure in Section 8.2.
128
-------
83 It is recommended that the laboratory adopt additional quality
assurance practices for «se 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 analysxs
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 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. 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
time 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 4 C for 24 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).
922 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
129
-------
TABLE 5. RECOMMENDED VOLUMES AND STORAGE at SAMPLES
Component
Collected Sample
Gravity Separation
Method
Sample for Resin
Column Concentration
Liquid/Liquid Extrac-
tion Method
Solids for Solid Waste
Extraction
Extracted/Concentrated/
Solvent Exchanged
Sample for Bioassay
Volume/Weight
Minimum of 30 L
Minimum of 30 L
10 L
3 L
500 g net weight
10 mL
Storage Conditions
V4 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 con-
tainer
4 C, amber glass vial
with Teflon cap liner
130
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I. SAMPLE INFORMATION
1. Sample No. Collection Date
(a)
2. Sampling Sitev
£. • LJ CLUL^f -L AL*^ w ^ w*- _ - - ' " —^—^^»-.» i
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. ' Sampling location
VUJLJ-Cl.1. J.U11 _--IUL/« _________________________———— . .
H ~~~ ~^ Sampling technique
Other
II. 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
Amber Glass Ambient 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
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 1 Environmental Assessment)
Biological Tests EPA-600/8-81-024, October, 1981. pp. 138.)
131
-------
of
3< mfthod TSsfM;38 <5VUSPended sol*ds 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 prefilter to the concentrator apparatus.
b. If the sample has >5% suspended solids by weight, the sample may
clfr'f her *6paf ted b? high-pressure filtration or high-speed 7
Th^I1^8 H°n nt° UqUld (<5% susPended «olids) and folids?
These two phases can be processed further by the Drinking Water
Protocol and Solid Waste Protocol, respectively? Urlnklng Water
c. If the sample has >5% suspended solids by weight, it may be
processed by a liquid/liquid extraction methof (Section^.*) . If
the bioassay from the sample recovered from this method is niga-
tive consideration should be given to processing a retained
liquid phase (10 L) by the method described in Section ™3.b.
9.3 Separatory Funnel Liquid-Liquid Extraction Method—
s.p.r»tory
wlth Blde-rmge PH
. 80Z
- r f^^
extractor and proceed as described in Section
132
-------
9.3.4 Add 100 mL of methylene chloride to ^^"^r
I-HP extraction procedure a second time, combining all extracts
ErLnmeyer flLr Perform another 100-mL extraction in the same manner.
9 3 5 Adlust the pH of the aqueous phases to less than 2 using the sulfurlc
rather! processing each separately through the remaining procedure.
the combined extract through a drying column contain! »8 at
will not flood with condensed solvent.
srs
.
tube with 1 to 2 mL of methylene chloride.
9.3.8 Concentrate the methylene chloride extract to 10 mL usi "8 'he nitrogen
by gas chTomatograph/mass spectrometer using Method 625 (44 *K z«. u
1979). Transfer the extract to a tared, 3-dram glass vial that is Jtched at
the DMSO extract should be recorded as mg organics/mL DMSO. Refrigerate
extract until ready for Ames testing.
939 If no aliquot was removed for analysis by gas chromatograph/mass
spectrometer, the DMSO extract represents a concentration factor of
133
-------
approximately 600x. If atl allquot was removed for
the DMSO
10.0 Calculations
134
-------
REFERENCES
Ames UN. McCann J, Yamasaki E. 1975. Methods for detecting carcinogens and
mutagens with the Salmonella/mammalian microsome mutagenicity test. Mutat.
Res. 31:347-364.
\
Brusick DJ, Young RR. 1981. IERL-RTP procedures manual: Level 1
environmental assessment biological tests. EPA-600/8-81-024. Litton
Bionetics, Inc. Kensington, MD. pp 138.
Committee 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. 44, No. 233. Fed. Reg. pp. 69548.
Hughes TJ, Pellizzari E, Little L, Sparacino C. Kolber A. 1980. Ambient air
pollutants: collection, chemical characterization and mutagenicity testing.
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 (OTIS 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 MW, Loper JC. 1980. Separation of mutagens from drinking water using
coupled bioassay/analytical fractionation. Int. J. Environ. Anal. Chem.
8:197-25.
U. 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: TIT Research Institute.
Williams LR, Preston JE. 1982. Standard procedures for conducting the
Salmonella/microsomal mutagenicity assay. U. S. Environmental Protection
Agency. EMSL.
135
-------
SECTION 5
NONAQUEOUS LIQUID WASTES
REVIEW OF THE LITERATURE
Introduction
Nonaqueous liquid environmental samples such as coal or petroleum
products and process effluents have been shown to contain mutagenic and cancer
causmg agents (Heuper 1953, Gradiski et al. 1983, Schultz et al 1983)
eSct oSf0th~term I038833" "^ been US6d t0 «»>««ct«i,. the advise''
use of thfsf biSLen °nTta m11XtUreS (Epl6r et a1' 1979)« To make -"I"
use of these bioassays, the samples must be prepared in such a way that thev
- atthey
ni! t0 !U bstantiate the hazard of nonaqueous liquid waste and
Th diffilt?" vtS,ha^ been hampered due t0 the ««Pl«ity of the problem.
SMDl" Is to £ % J ^ natUre °f analysis» ln tha< (a) a nonaqueous
SX«, of rt ? ^ ^ aqUSOUS SyStem' (b) inhlbitory and/or synergistic
nfn^! C?TP 6X muixtures and toxicity of one or more particular com-
foTllc* r ,C07 TtG the resultin8 interpretation and (c) limitations exist
for each test for detecting all types of carcinogens; for example, certain
components in the unknown sample can be negative in one system and mutagenic
rSm dlfferent metab°11C activat*°" capiilitSs or
targets.
«oHHN°nTeOUS ^qUld envlronmental samples are those containing less than 5%
solids. The sample can contain mixtures of volatile (b.p. 36 C to 100 C)
moderately volatile (b.p. 100 C to 300 C) and nonvolatile (b p. >300 C)
organic compounds In addition, the samples are generally entities of poorly
defined and possibly continuously changing chemical composition.
The method for sample preparation can be a simple one, such as evapora-
scheme° ^hTtlT? "T^ " * COmPllcated« deta"ed fractionation
scheme. The technique used most successfully to date is to separate the
constituents of the mixture into a manageable number of chemically distinct
fractions Each fraction is then bioassayed to determine whether mutagenic
1 **' 'T "" ^^ indePend^t of synergism, toxic inter-
, er-
of wid.l ff COmPlicatlons as a "suit of the interaction of constituents
onsu
srt Pr°Pe«ies (Guerin et al. 1978, 1982). The methods of .
sellrltir 3H co^^ce of the chemical nature of the compounds to be
separated and, more specifically, of differences in physical properties
(boiling point, solubility, etc.), polarity and acidl/basic characteristics
136
-------
However the primary requirement is that all of the constituents present in
thTTtarting material be recovered in the fractions to be bioassayed.
The methods for preparation of complex mixtures *°* .%
procedures should be simple and cost-effective.
Several methods have been developed to prepare nonaqueous liquid environ-
Ames Salmonella assay is established.
Summary of Currently Available Methods
The available literature on the preparation of nonaqueous li^,6s for
fication or active mutagens^ecomes more realistic. Although our Pjo-ty
objective is to evaluate the mutagenic potential of nonaqueous liju ids, the
literature pertaining to chemical identification is quite relevant to the
preparation of fractions for mutagenicity screening.
Guerin et al. (1980) evaluated the mutagenic potential of petroleum
substitute using a chemical class fractionation coupled with the standard Ames
eft A Iiquid8sample was evaporated at 40 C under water "P*"^; f ^f *
"
ri.
between ether and IN HGl! Alkaline constituents of the sample concentrated
the "idfc aquLus phase, whereas acid and neutral ^"^^^S
in the organic phase. Subsequent mixing of the organic phase with IN NaOH
preferentLlly extracted the acidic constituents. The constituents of the
was achieved by using tetrahydrofuran and isopropanol. The gel chromato-
graphiclrocedure thus produced the following fractions: ^ydrophilic, poly
meric, hydrogen bonding, aliphatic, simple aromatic and polyaromatic.
137
-------
.. i
et al. (1980, 1981); TimourlL et a ?198«
Schoeny et al. (1981) aonHpH
tion Jth organic'soL^ M
gasification and liquefaction
yield mutagenic
SS
of
rd by Rubln et
? 7 ^ PetGrSen (19?9)' Ho
°Ste and Sklarew (1982).
-rial extrac-
from coal
d"
h^d^t
°±1S ^ Dissolving the
.
polynuclear aromatic hydrocarbons
the
slurry. However
DMSO
of
or DMSO to extract oil sa»ples
remits that we« craMe ^th tho«
findings suggest that the "
achieved bylhe detergent
extraction efficiency?
SOlUb"1Zl"8 aSentS
with pure BP in DMSO. These
1 °f '£" ^'"^ "" ""
eliminating the concern as to
in
(a) Registered trademark.
138
-------
organic components in the sample are mutagens detected either in the plate
incorporation assay or in the preincubation assay, a gravimetric (GRAY)
analysis is performed (Lentzen et al. 1978, Section 9.4.2). Briefly, by this
simple procedure, an aliquot of the sample is evaporated in a preweighed
aluminum weighing pan and then dried to a constant weight. The residue weight
is determined, and the GRAV nonvolatile organic content is calculated from the
residue weight and reported as one number for the whole sample. At least
10 mg of sample residue should be weighed, but no more than 10X of the sample
should be used for GRAV analysis. Highly viscous liquids and pastes are
weighed directly, placed in a suitable solvent and dosed on a weight-per-
volume basis.
From the above literature reports, we conclude that the best way to^
evaluate the initial mutagenicity of nonaqueous liquids is to test them neat
or as DMSO suspensions or solutions. If no mutagenic activity is found, acid,
base and neutral partitioning should be performed to fully evaluate the
mutagenic potential of the sample.
139
-------
REFERENCES
Ames BN, McCann J, Yamasaki E. 1975. Methods for detecting carcinogens
Mutat? Rfs!%T;347!S64Salm0nella-/mammalian"miCrOSOme mutagenlclty test.
Amf1 QT^KT1Q7QTj4.j£» ^
cancer!" Science ^m^l™*00™**1 c^c.!s .causing mutations and
Baden JM, Kelley M, Whorton RS et al. 1977. Mutagenicity of halogenated
ether anesthetics. Anesthesiology 46:346-350. naiogenated
EPA. 1982. Ames Mutagenicity Assay. Organic Extraction Procedure for
Denver! co!"01"11611'31 Samples' N^ional Environmental Investigations Center,
pollunt , *** ^ AnalySlS °f ^ract.ble Organic
Epl-fiJo/4 ftl nm in,o?do^rial and MunlclPal Wastewater Treatment Sludge.
Ss VeasT NV PP' 3* Environmental Monitoring Systems Laboratory,
Epler JR, Young JA> Hardigree AA et al. 1978. Analytical and biological
analysis of test materials from the synthetic fuel technologies. I.
^1 •
testinf ofRah f ' Grrl", m' 1979' Evaluation of feasibility of mutagenic
30: 179^184 ° products and effluents. Environ. Health Perspect.
Gradiski D Vinot J, Zissu D, Linasset JC, Lafontaine M. 1983. The
on the skin of mice.
?R> H° CH' Epl6r JL' Ra° TK" 1978« Short-term bioassays of
Bioassavsh 'XtUreS' Part X« Chemistry. In: Application of Short-term
££-600/9 78 S? 9?oa^r ^ Analysis of Co-plex Environmental Mixtures.
Trtangfe ^Irk NC.PP' ' HGalth Eff6CtS ReS6arCh Lab-^ory, Research
Guerin MR Ho CH, Rao TK, Clark BR, Epler JL. 1980. Polycyclic aromatic
Environ. ^™\$* ££****** ^^ ^^ ln »<*™1*™ substitutes.
Guerin MR, Rubin TB, Rao TK, Clark BR, Epler JL. 1982. Distribution of
mutagenic activity in petroleum and petroleum substitutes. Fuel 60:282288.
140
-------
Haworth S, Lawlor T, MortelmatiB K, Speck W, Zeiger E. 1983, Salmonella
mutagenicity test results for 250 chemicals. Environ. Mutagen. Supplement
1:3-142.
Hermann M, Chande 0, Weill N, Bedonelle H, Hofnung M. 1980, Adaptation of
the Salmonella/mammalian microsome test to the determination of the mutagenic
properties of mineral oils. Mutat. Res. 77:327-339.
Heuper WC. 1953. Experimental studies on carcinogenesis of synthetic liquid
fuels and petroleum substitutes. Arch. Ind. Hyg. Occup. Med. 8:307-327.
Ho CH, Ma CY, Clark BR, Guerin MR, Rao TK, Epler JL. x 1980. Separation of
neutral nitrogen compounds from synthetic crude oils for biological testing.
Environ. Res. 22:412-422.
Ho CH, Guerin MR, Clark BR, Rao TK, Epler JL. 1981. Isolation of alkaline
mutagens from complex mixtures. J. Anal. Toxicol. 5:143-147.
Jones AR, Guerin MR, Clark BR. 1977. Preparative-scale liquid chroma-
tographic fractionation of crude oils derived from coal and shale. Anal.
Chem. 49:1766-1771.
Lentzen DE et al 1978. In: IERL-RTP Procedures Manual: level 1
environmental assent (second edition). EPA-600/7-78-201 (NTIS PB 293795),
279 pp. Research Triangle Institute, Research Triangle Park, NC.
Ma CY, Ho CH, Quincy RB et al. 1983. Preparation of oils for bacterial
mutagenicity testing. Mutat. Res. 118:15-24.
Matsuoka A, Shudo K, Saito Y, Sofuni T, Ishidate Jr. M. 1982. Clastogenic
potential of heavy oil extracts and some aza-arenes in Chinese hamster cells
in culture. Mutat. Res. 1-2:275-283.
Pelroy PA, Petersen MR. 1979. Use of Ames test in evaluation of shale oil
fractions. Environ. Health Perspect. 30:191-203.
Rubin IB, Guerin MR, Hardigree AA, Epler JL. 1976. Fractionation of
synthetic crude oils from coal for biological testing. Environ. Res.
12:358-365.
Schoeny R, Warshawsky D, Hollingsworth L, Hund M, Moore G. 1981.
Mutagenicity of products from coal gasification and liquefaction in the
Salmonella/microsome assay. Environ. Mutagen. 3:181-195.
Schultz TW, Dumont JN, Buchanan MV. 1983. Toxic and teratogenic effects of
chemical class fractions of a coal-gasification electrostatic precipitation
tar. Toxicology 29:87-99.
Simmon VF, Kauhanen K, Tardiff RG. 1977. Mutagenic activities of
chemicals identified in drinking water. In: Progress in Genetic
Toxicology, pp.249-258. Elsevier/North Holland Biomedical Press, NY.
141
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J. Chrogr
M2
-------
NONAQUEOUS LIQUID WASTES WORKGROUP REPORT
Workgroup Tasks
Summary of Tasks—
In summary, nine tasks were identified during the general sessions and
workgroup discussion. These nine tasks are listed under the Summary of
Workgroup Progress.
Key Peer Review Comments—
There were no unresolved written peer review comments from the
pre-meeting review.
Summary of Workgroup Progress—
a. Definition - Nonaqueous liquid wastes are those liquids whose
major component is not water, including water-soluble organic
liquids such as acetone from an industrial process, water-
insoluble liquids such as dichloromethane from a similar
industrial process and liquids that might be composed of light to
heavy oils.
b. Storage - The samples, regardless of source, should be stored in
Teflon-capped amber-glass bottles with zero headspace or argon
blanket, whichever is applicable, at 4 C or lower. Aliquots, such
as 10 ml per vial, should be prepared before storage and
transportation. The neat material should be tested as soon as
possible.
c. Water-soluble compounds - This is a non-issue because we will use
organic solvents for extraction, and these compounds will be
extracted.
d. Solvent - We recommend the use of dichloromethane because it is a
universal solvent and has been applied to most other media types
and because it is easy to obtain high purity dichloromethane.
Note: We recommend that a research project be done to compare
CH-C1 and diethyl either for extraction efficiency.
e. pH - We will test the neat sample as is, without adjustment of pH.
For fractionation, we suggest extracting bases with IN HC1 first,
and then separating the acids and neutral fraction.
f. Blank - (1) For the Ames test, solvent control and positive
controls are necessary for all three assays to be performed.
143
-------
2 res'iduers^^ ^t** *?* USlng the f«ctian«tlon scheme.
residues should be subjected to the Ames test.
fcnmm «. - ~ We Su88est spiking half of the sample with
known mutagens. Some possible standards could be ben2o(a)pyrene
» nitroh" rta8^S; 2-an.inoanthracene for base mutagens and
p-nitrobenzoic acid for acid mutagens.
°f each fraction and the original sample
Should
ine tractions should be reconstituted and assayed together with
each fraction to evaluate additive/synergi^tic/inhibftory effects.
i. Vapor phase organics and modification of the Ames test - We will
use the desiccator assay to detect the vapor phase organic
™utflgcns•
Summary of Key Workgroup Discussions
Major Consensus Opinions—
of opinions is that the proposed protocol is sound.
were not delineated in the original protocol have been
Unresolvable Issues—
There are no unresolved issues.
Evaluation of Proposed Protocol
Adequacy—
liquids^ Pr°tOCo1 1S belleved to be adequate for the majority of nonaqueous
Validity-
water I!l«n?»MOC01 SlTld be validated in a range of water-soluble and
water-insoluble organic matrices.
Limitations—
a. There is the potential for alteration of the mutagenic activity
using an acid-base extraction fractionation procedure.
b. If toxicity is observed in the DMSO-acids, bases or neutrals
fractions, we are not recommending further fractionation.
c. Low concentrations of highly polar mutagens may not be detected as
a result of the fractionation procedures.
Comparison to the Other Five Media Protocols—
The fractionation procedure should be identical to those in the soil and
solid wastes protocols if HPLC methods are not adopted.
144
-------
Other Data or Information Requirements
Information Gaps—
Data are needed for the selection of an optimum extraction solvent and
sample storage condition.
Research Program Needs—
(a) No data on other liquids besides oils. (b) Chromatographic methods
for sample fractionation should be studied.
145
-------
PREPARATION OF NONAQUEOUS LIQUID WASTES FOR MUTAGENICITY
1-° Scope and Application
srss^i
I £1 v yS^MftrswE.:?^ ^ -
H™! dime'hylsulfo*lde OW80) and are stable to acid base solvent partitioning
However, the user is cautioned that not all chemicals are stable through this
r °r eXample' benzidine «« *• s^ect to oxidation losseslurin
roug t
h °r eXample' benzidine «« *• s^ect to oxidation lossesluring
the solvent concentration; oc-BHC, 6-BHC, endosulfan I and II and end r in are
subject to decomposition under the alkaline conditions of the extraction step
i1 -
1.4
mu
taeenic?tvneat nter.M\nA the flnal e^"cts are subjected to the Ames
tagenicity assay using the procedures of Williams and Preston (1983).
2.0 Summary of Method
v .u *T° determine the mutagenic potential of nonaqueous liquids as measured
recommend f^onella/mammalian-enzyme assay, the following protocol is
recommended (also diagrammed in Figure 13) for the sample preparation. In
Step l the desiccator assay is performed on the neat material. The desiccator
th^t'are o^en'mi'^rf °\°f T^* ^^ (8UCh 3S Chlorinated "iv
that are often missed in the plate incorporation and preincubation assays
146
-------
If volume is reduced
by >90%, repeat these tests,
redissolve residue in DMSO
Preincubation Assay
Plate Incorporation Assay
Desiccator Assay
Step 1
I
I Sample [
Step 2
1. Remove volatiles by rotary
evaporation at 40 C
Concentrate
Step 3
1. Partition sample into dichloromethane
2. Extract sample with W_ HC1
AQ1
01
F Aqueous Fraction I
Organic Fraction
1
. Basify to pH 11 with
IN NaOH
2. Extract with dichloro-
methane
[ Organic Fraction | JAqueous Fraction j
Bases
I 1. Rotary evaporate to a
I residue at 40 C
1. Extract with IN NaOH
AQ2
02
t2. Weigh residue JAqueous Fraction I
3. Dissolve residue in DMSO 1 | '
DMSO-Bases Fractii
Lon |
I Organ
ic Fraction
[03
1. Adjust pH to 2 with Neutrals
IS^ HC1 I 1. Rotary-evaporate to
2. Extract with dichlororoethane I a residue at 40 C
12. Weigh residue
I 3. Dissolve residue in DMSO
| Organic FractionH \ A""eous FractionJ ^MSO-Neutrals Fraction
Acids
11. Rotary evaporate to a
residue at 40 C
2. Weigh residue
T. Dissolve residue in DMSO
DMSO-Acids Fraction
Each DMSO fraction from Step 3 will be bioassayed.
Figure 13, Sample preparation scheme for bioassay.
147
-------
not effectively concentrated by the evaporaion prcer or produce s
53r?IS?-s' - s^-srsjis a-
all samples should be handled identically.
with IN HClSte?h!'flthe llqfd ls disflved ^ dichloromethane and extracted
fl
xtracd ith ? MU Jayer ^^ 1S adJusted to PH 11 with IN NaOH and
extracted with dichloromethane to yield the basic fraction. This fraction is
dried over anhydrous sodium sulfate, filtered and rotary-evaporated at 40 C to
rsl- is weighed
1 i t is
become the DMSO . ^l" *? that ^ the °3 fraCti°n and ls --k-d «P to
-±^^h^^^
that areoxiror1^1^ T^ ±S Preferred because it isolates components
8 °therwise interfere with the assay. Thus, the
Donl f. u .
She distributing I" ^%con,trlbutors to mutagenicity is increased.
the distribution of activity in the various fractions provides a preliminary
6 t °f ehemlcals responsible for the mutagenici?y? Further-
148
-------
3.0 Definitions
Nonaqueous liquid waste environmental samples are those liquids whose
major component is not water, including water-soluble organic liquids (such as
acetone), water-insoluble liquids (such as dichloromethane) or liquids that
might be composed of light to heavy oils (such as coal or petroleum products
or process effluents). The samples contain less than 5% solids and may
contain mixtures of volatile (b.p. 36 C to 100 C), moderately volatile (b.p.
100 C to 300 C) and nonvolatile (b.p. 300 C) organic compounds. In addition,
the samples are generally entities of poorly defined and possibly continuously
changing chemical composition.
4.0 Interferences
4.1 Method interferences may be caused by contaminants in solvents,
reagents, glassware and other sample processing hardware that lead to discrete
artifacts. All of these materials must be routinely demonstrated to be free •
from interferences under the conditions of the analysis by running laboratory
reagent blanks.
4.1.1 Glassware must be scrupulously cleaned. All glassware should be
cleaned as soon as possible after use by rinsing with the last solvent used in
it. This should be followed by detergent washing with hot water and rinses
with tap water and reagent water. It should then be drained dry and heated in
a muffle furnace at 400 C for 15 to 30 min. Some thermally stable materials,
such as PCBs, may not be eliminated by this treatment. Solvent rinses with
acetone and pesticide-quality dichloromethane may be substituted for the
muffle furnace heating. Volumetric glassware should not be heated in a muffle
furnace. After drying and cooling, glassware should be sealed and stored in a
clean environment to prevent any accumulation of dust or other contaminants.
It should be stored inverted or capped with aluminum foil.
4.1.2 The use of high purity reagents and solvents helps to minimize
interference problems. Purification of solvents by distillation in all-glass
systems may be required.
4.2 Matrix interferences may be caused by contaminants that are
coextracted from the sample. The extent of matrix interferences will vary
considerably from source to source, depending upon the nature and diversity of
the system being sampled.
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. The laboratory is responsible for maintaining a current awareness
file of OSHA regulations regarding the safe handling of the chemicals specified
in this method. A reference file of material data handling sheets should also
be made available to all personnel involved in the chemical and mutagenicity
analyses.
149
-------
5.2 An example of safety procedures is described in detail as follows-
,
150
-------
Work with a test article is performed on disposable, plastic-backed
absorbent pads within Baker Class II, Type B hoods (NCB-4 or NCB-6). The
exhaust air is filtered by a HEPA filter in the hood and by charcoal filters
on the roof. Hood surfaces are wiped with ethanol and disinfectant before and
after completion of an experiment. The hoods are inspected and certified once
a year by an outside testing agency. The charcoal filters are tested for
effectiveness every week by laboratory personnel. All hoods are equipped with
warning devices to indicate insufficient air flow during a power failure or
because of a mechanical or other failure.
Laboratory personnel retrieve the secondary container with the
requested amount of test article from the pass-though^ window of the chemical
repository. All further manipulations of the test article are conducted in
the hoods. Any chemical solutions remaining after completion of the test are
disposed of in properly labeled 1-gal plastic containers.
Spill packs containing absorbent material and other items for cleaning
up minor spills are available in the regulated areas. All laboratories are
equipped with fire extinguishers, first-aid kits, hand-held eye washers and
telephones. Emergency numbers to be called and signs outlining the correct
procedures to follow in case of an emergency are posted in all laboratories.
The laboratory should comply with the applicable state and federal
Occupational Safety and Health Administration regulations and with federal
security and safety requirements specified under the Williams-Steiger
Occupational Safety and Health Act of 1970. The laboratory should also follow
the National Cancer Institute Safety Standards for Research Involving Chemical
Carcinogens, DHHS Publication No. NIH-76900.
6.0 Apparatus and Equipment
All specifications and brand names are suggestions only.
6.1 All the equipment and materials required for the Ames bioassay
(Williams and Preston 1983).
6.2 Desiccator - 9 L, all glass, with lid and porcelain platform.
6.3 Glass petri dishes - 15 x 100 mm.
6.4 Disposable glass pipette - 1, 5 and 10 mL.
6.5 Graduated cylinder - 100 or 250 mL.
6.6 Vials - Amber glass, with Teflon-lined screw caps.
6.7 Sample bottles - 1,000-mL to 5,000-mL glass with Teflon-lined screw
caps.
6.8 Beakers - 100 and 250 mL.
6.9 Flasks - Erlenmeyer, 250 mL.
151
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6.10 Separatory funnels - 250 mL with Teflon stopcocks.
6.11 Rotary evaporator - Buchi or equivalent.
6.11.1 Flasks - Round bottom, 50, 125, 250 and 500 mL.
6.11.2 Traps - Glass.
6.11.3 Clamps.
6.11.4 Water bath - Heated with temperature control (±2 C).
6.12 Balance - Analytical, capable of accurately weighing 0.0001 g.
6.13 Nitrogen evaporator - Equipped with Teflon or glass jets and
temperature control, Meyer N-evap Model 112 or equivalent.
6.14 Ultrasonic Bath - Bransonic 12 or equivalent.
7-0 Reagents and Consumable Materials
7.1 Acetone - "Distilled in Glass" or equivalent, stored in original
containers and used as received. ±&±w.
7.2 Dichloromethane - "Distilled in Glass" or equivalent, stored in
original containers and used as received.
7.3 Dimethysulfoxide - High purity, supplied by Pierce or equivalent,
stored in original containers and used as received. Should be stored under
nitrogen or argon when opened.
7.4 Reagent water - Reagent water is defined as a water in which an
interference is not observed at the method detection limit of each parameter
or interest.
7.5 Hydrochloric acid solution (IN) - Slowly add 8.3 mL of HC1 (12N) to
reagent water and dilute to 100 mL.
7.6 Sodium hydroxide solution (IN) - Dissolve 4 g NaOH in reagent water
and dilute to 100 mL.
7.7 Sodium sulfate (Na SO ) - Anhydrous, granular. Clean by overnight
Soxhlet extraction with diShloVomethane, drying in an oven at 110 to 160 C and
then heating to 650 C for 2 h. Store in a glass jar tightly sealed with
Teflon-lined screw cap.
7.8 Nitrogen gas - Cylinder, secured and grounded.
7.9 Argon gas - Cylinder, secured and grounded.
8'° Sample Collection. Preservation and Handling
152
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8.1 Grab samples must be collected in glass containers. The containers
should be prewashed with high purity acetone and dried before use. A sample
collection form, as illustrated in Figure 14, should be reported on each
sample. Aliquots, such as 10 mL per vial, should be prepared before storage
and transportation. The samples, regardless of source, must be stored in
Teflon-capped amber glass bottles with zero headspace or under a blanket of
inert gas (e.g., argon). The samples must be iced or refrigerated at 4 C or
below from the time of collection until preparation.
8.2 All sample preparation and testing procedures must be performed under
yellow/gold lighting or equivalent to avoid photoactivation or decomposition
of the product. v
8.3 Samples can be warmed to room temperature over a 1- to 2-h period
prior to testing. If the samples are to be reused, they should be purged with
argon gas, sealed and stored at 4 C or below.
8.4 All samples should be tested and processed as soon as possible.
9.0 Calibration and Quality Control
9.1 Each laboratory that uses this method is required to operate a formal
quality control program. The minimum requirements of 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 must be compared with established performance
criteria to determine if the results of analyses are within accuracy and
precision limits expected of the method.
9.2 To establish the ability to generate acceptable accuracy and
precision, the analyst must perform the following operations with spiked and
solvent blank samples.
9.2.1 Surrogate spikes - The laboratory is required to spike all of their
samples with the surrogate standard spiking solution to monitor spike
recoveries. Suggested surrogate compounds are 2-aminoanthracene,
benzo(a)pyrene and p-nitrobenzoic acid. If the recovery for any surrogate
standard does not fall within the control limits for method performance, the
results reported for that sample must be qualified with respect to the total
mutagenicity recovered.
The concentration of the surrogate mutagen should exhibit a response
that is at least five times the background spontaneous mutation rate at the
highest dose in the dose-response curve. The spiked sample dose-response
curve is then compared to the standard curve for total mutagenicity recovered.
9.2.2 Method blank - Before processing any samples, the analyst should
demonstrate, through the analysis of an 80-mL aliquot of reagent water, that
all glassware and reagents are interference free. Each time a set of samples
is extracted or there is a change in reagents, a laboratory reagent blank
should be processed as a safeguard against laboratory contamination.
153
-------
II.
1.
2.
SAMHJ^JjlFORMAT ION
Sampling Site
Field Samp 1 ing Manager " (on-lit^)
r!nnt-T-ar.<-«»- '
EPA Project~0ffi^
Source Sampled
Quantity Sampled/Unit^
O n*w_. "!_»•* .
. Slurry. solid.
Other InTo^mation as
Sampling location
j»i « .
Sampling technique
HANDLING & SHIPPTMC
Describe Sample Treatment Prior to
pv^a"*—*••}, stored
Field Storage and Shipping Conditions
Amber Glass
Other"
Utner
Ambient'
p6" r±86rate (0 to
Freeze (-20 C)
Dry Ice
C>
Shield from light
Mode and Carrier for Shipping
Comments
(This form should be c
each sample. A CODV should
(Extracted from-
S
-------
10.0 Procedure
10.1 Sample Preparation Procedure--
Step l.A Desiccator Assay (Figure 15)
1. Pour an aliquot of the nonaqueous liquid into a standard glass
petri dish to cover the bottom of the dish.
2. Test liquids for mutagenicity following the Desiccator Assay
Protocol as described in Section 10.2.3.
\
Step l.B Plate Incorporation and Preincubation Assay
1. Dissolve or suspend the test liquid in DMSO at a concentration of
20 mg/mL. For suspending the liquid, use an ultrasonic bath at
room temperature for 5 min.
2. Test the solution or suspension using the plate incorporation and
preincubation protocols as described in Interim Procedures for
Conducting the Salmonella/Microsomal Mutagenicity Assay (Ames Test)
(EPA Report-600/4-82-068, Williams and Preston, 1983).
3. If toxicity is observed, perform 10-, 100- and 1,000-fold
dilutions of the neat liquid in DMSO until a dose-response or
background (negative) response is achieved.
Step 2. Concentration
1. If the test liquid possesses a low viscosity, weigh an aliquot
g) in a tared round-bottom flask.
2. Concentrate the liquid by rotary evaporation at 40 C, using a
Buchi rotary evaporator or equivalent with water aspiration or
equivalent .
3. Weigh the residue. If a 10-fold concentration factor is achieved,
retest the residue in the plate incorporation and preincubation
assays after dissolving or suspending the residue in a minimum
amount of DMSO.
Step 3. Fractionation
If a toxic or negative response is observed in Steps 1 or 2, the test
liquid is fractionated by acid-base partitioning as described below.
1. Weigh approximately 5 g of the test liquid into a 250-tnL
Erlemmeyer flask and add 100 mL of high purity dichloromethane.
2. Swirl the flask until the liquid dissolves. If solution is not
complete after 10 min, add a magnetic stir-bar and stir the
solution on a stir-plate for 1 h. If solution is not complete,
155
-------
Porcelain ^
Shelf
Fan
1 1 r • .
;==^
V " " j1 " " " " " ii ii -i
V i ' "If/
\ /
A r»— *^-^_— — i /
3 T.
g
-X
^ Petri Plates
with Salmonella
Glass Petri
*• Plate for
Test Chemical
^ Magnetic
Stirrer
Figure 15. Desiccator assay.
-------
separate the layers using a separatory funnel, and retest the
insoluble liquid as described in Step 1 plate incorporation and
preincubation assays.
3. The dichloromethane phase is transferred to a 250-mL separatory
funnel and extracted with 3 x 75 mL portions of IN HC1. Save the
aqueous (Step 3.4) and organic phases (Step 3.6).
4. The aqueous HC1 phase is basified with IN NaOH to pH 11 and
extracted with 3 x 50 mL portions of dichloromethane. The
dichloromethane extracts are combined, dried over anhydrous sodium
sulfate, filtered and rotary evaporated at: 40 C to approximately
4 mL. The dichloromethane concentrate is transferred to a 8-dram
glass vial with a Teflon-lined cap with a minimum amount of fresh
dichloromethane and blown dry with a gentle stream of nitrogen
gas.
5. The residue is capped, weighed and dissolved in a minimum amount
of DMSO. The vial is labeled with the sample identification, the
concentration and "contains the DMSO-bases."
6. The dichloromethane phase from Step 3 is extracted with 3 x 75 mL
port-ions of IN NaOH in a 250-mL separatory funnel. Save the
aqueous and dichloromethane phases.
7. The dichloromethane phase is dried over anhydrous sodium sulfate,
filtered and concentrated to approximately 4 mL by rotary
evaporation* The concentrate is transferred to a tared 8-dram
vial with a minimum amount of fresh dichloromethane and blown dry
with a gentle stream of nitrogen gas.
8. The residue is capped, weighed and dissolved in the minimum amount
of DMSO. The vial is labeled with the sample identification, the
concentration and "contains the DMSO-neutrals."
9. The aqueous phase from Step 6 is carefully acidified with IN HC1
to pH 2 in a 500-mL separatory funnel. The solution is then
extracted with 3 x 100 mL portions of dichloromethane.
10. The dichloromethane extracts are combined, dried over anhydrous
sodium sulfate, filtered and concentrated to approximately 4 mL.
The concentrate is transferred to a tared 8-dram glass vial and
blown dry with a gentle stream of nitrogen gas.
11. The residue is capped, weighed and dissolved in a minimum amount
of DMSO. The vial is labeled with the sample identification, the
concentration and "contains the DMSO-acids."
12. From the weight of the residues and the initial sample weight, the
percent distribution of weight into the DMSO-bases, DMSO-neutrals
and DMSO-acids fractions is calculated. Also, the percent
157
-------
recovery of total mass is determined as the sum of the acid base
and neutral fraction weight divided by the initial sample weight
10.2 The Ames Bioassay Procedure —
s.rsi-.s:
-
COn't-laln« °'6* NaC1» ^.05 mM biotin and
2. 0.10 mL of indicator organisms (about 108 bacteria)
3. 0.50 mL of metabolic activation mixture (if appropriate)
4. Appropriate amount of the test article (e.g., 20 mg/mL)
ss
=
•'
.
158
-------
1 Duplicate plates for each of the five strains without or with one
activation system (*10 plates per desiccator per dose level).
2 Duplicate plates for one strain without and with up to four
activation systems (*10 plates per desiccator per dose level).
A total of five dose levels are used, in addition to the negative
solvent control. The positive control chemicals are tested in the standard
plate incorporation and preincubation assays.
For volatile liquids, the Salmonella plates are prepared as described
for the assays in agar, but no test chemical is added., The plates, with the
lids having been removed, are placed side by side on a perforated shelf in a
9-L desiccator. A known volume, such as 2, 5 and 10 ml of the test chemical,
is added to a glass petri plate, which is then placed in the center of and
attached to the bottom of the shelf. The desiccator is sealed and placed on a
magnetic stir-plate in a room maintained at 37 C. A magnetic stirrer with
vanes, placed in the base of each desiccator, ensures adequate dispersion of
the chemical. After incubation for a known period, usually 8 h, the plates
are removed from the desiccators, their lids are replaced and they are
incubated at 37 C for an additional period up to 48 h. The number of his+
revertants is counted and recorded. The desiccator assay is illustrated on
the next page. After each experiment, the desiccator is vented in a chemical
fume hood overnight and then cleaned with soap and bactericide, followed by
hexane and ethanol rinse.
11.0 Calculations
The calculations required for this protocol are the determinations of
weight loss due to rotary evaporation, the percent of the initial sample
weight partitioned into the acids, bases and neutrals fractions and the total
recoverable weight resulting from fractionation.
Consider the following definitions:
A = Initial sample weight
B = Sample weight after rotary evaporation
C = Base fraction residue weight
D = Acid fraction residue weight
E = Neutral fraction residue weight
Then:
Weight loss due to Rotary Evaporation = A-B
% Weight loss due to Rotary Evaporation = (A-B/A) x 100
% Weight of sample partitioned into base fraction « (C/A) x 100
% Weight of sample partitioned into acid fraction = (D/A) x 100
% Weight of sample partitioned into neutral fraction = E/A x 100
% Total recoverable weight - ((C + D + E)/A) x 100
A data report sheet is illustrated in Figure 16.
159
-------
Sample Identification No.
Date:
Analyst:
Volume removed for Desiccator Assay: v
Weight Removed for Plate Incorporation and Preincubation Assays:
% Weight Loss from Rotary Evaporation:
Fractionation Sample Weight:
Base Residue Weight
Acid Residue Weight
Neutrals Residue Weight
Total Recovery: BASgg_+_.ACIDS + NEUTRALS
SAMPLE WEIGHT x 100 -
Figure 16. Data report sheet.
160
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REFERENCES
Ames BN, McCann J, Yamasaki E. 1975. Methods for detecting carcinogens
and mutagens with the Salmonella/mammalian-microsome mutagenicity test.
Mutat. Res. 31:347-364.
Baden JM, Kelley M, Whorton RS et al. 1977. Mutagenicity of halogenated
ether anesthetics. Anesthesiology 46:346-350.
EPA. 1982. Ames Mutagenicity Assay. Organic Extraction Procedure for
Aqueous Environmental Samples, National Environmental Investigations Center,
Denver, CO.
EPA. 1984. Method 625-S, Protocol for the Analysis of Extractable Organic
Priority Pollutants in Industrial and Municipal Wastewater Treatment Sludge.
EPA-600/4-84-001, pp. 234-273. Environmental Monitoring Systems Laboratory,
Las Vegas, NV.
Haworth S, Lawlor T, Mortelmans K, Speck W, Zeiger E. 1983. Salmonella
mutagenicity test results for 250 chemicals. Environ. Mutagen. Supplement
1:3-142.
Maron DM, Ames BN. 1983. Revised methods for the Salmonella mutagenicity
test. Mutat. Res. 113:173-215. . .
Selby C, Calkins J, Enoch H, 1983. Detection of photomutagens in natural and
synthetic fuels. Mutat. Res. 124:53-60.
Simmons VF, Kauhanen K, Tardiff RG. 1977. Mutagenic activities of chemicals
identified in drinking water. In: Progress in Genetic Toxicology,
pp.249-258. Elsevier/North Holland Biomedical Press, NY.
Wang YY, Rappaport SM, Sawyer RF, Talcott RE, Wei ET. 1978. Direct-acting
mutagens in automobile exhaust. Cancer Lett. 5:39-47.
Williams LR, Preston JE. 1983. Interim Procedures for Conducting the
Samonella/Microsomal Mutagenicity Assay (Ames Test). EPA-600/4-82-068.
Environmental Monitoring Systems Laboratory, Las Vegas, NV.
Yahagi T, Degawa M, Seino Y et al. 1975. Mutagenicity of carcinogenic azo
dyes and their derivatives. Cancer Lett. 1:91-97.
161
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SECTION 6
SOILS AND SEDIMENTS
REVIEW OF THE LITERATURE
Introduction
Soil is the unconsolidated material on the earth's crust capable of
° "" hat haS Se"led t0 the b°"°m oToXs
l b°"°m oos been
by, a liquid is described as a sediment Fertile
t T r - < STSS. -
protocols have been developed specifically for the preparation of soil and
--
.
the preparation of soil and sediment samples for biological testing?
nr.* *here 1S v"y little information available describing studies where
organxc compounds from soil or sediment samples have been extracted for
ar
h* S Sy8tem- ere haVe bee" a large -mber
however, involving the extraction of soil humic acid
fr«rM«
carbons fi . lltTS* **?*** prlmarlly —crib., the extraction of hydro-
SI?™««.« A f11^3'10 a"d a™»atic compounds) from contaminated soils or
bioassavs'to oh7 "^ th\l3St f°Ur yearS ha8 research been conducted using
bioassays to characterize the mutagenic potential of soil (Goggelmann and
ill »"SI»«f.rt?r<7oi?Mi DTeUy " a1' 1983) ^ BedimSt (Snae ft .1.
iysi, Hirayama et al. 1981, Suzuki et al. 1982, Sato et al. 1983).
assavs to I!T J I chemical analysis employs short-term mutagenicity
component* nf mutf8enl,c fract*°»s and to interpret the interactions of the
components of a complex mixture; chemical analysis is used to characterize
cofi™ rStltUentS ^ t0 Verlfy the absence °f Artifacts generated in the
cox J.6C t ion or extract Ion m-npoac TK^ 01^4^.1^^^ i * « . 4
..*«« j—, ,^i ^ process. This biological and chemical characteri-
. inciuaes tnree major phases: (a) chemical extraction to
compounds present and separate them from other associated
re 17a); (b) chemical fractionation using techniques for sepa-
basic and neutral organic compounds (Figure 17b); (c) final
identif1r*MnaC f fra"ions b7 chromatographic techniques and, if possible,
(Figurf 17cK sPeciflc "tugenic compounds by mass spectral techniques
162
-------
Extract
with
Ext met
witli
Acid
Acidify*
Extract
with
Solvent
Aqueous mf Discard
B. ClH-nil.iil Frai't ionatlim Scheme
(a) Sample size
full identification.
C. Chemical Isolation Scheme
Figure 17. Chemical procedures, bioassay-directed chemical analysis.
-------
Extraction
sedin , °f comP°unds m*y be solvent extracted from soil and
previouslv ^ ?J J"?8 ?* Varl°US laboratory techniques which have been
samples yAUhou^ S 5& *f raCtlon of industrial wastes or environmental
samples. Although the Soxhlet extraction is the procedure most frequentlv
used Donnelly and Brown (1981) compared the Soxhiet extraction with a more
rapid extraction using a blender and found no appreciable difference in the
mutagenic potential of the extracted material using either method
that Ire readJl* I?*""??? *! dt?lgned for use with finely-divided solids
I? „ ?, e ly dlsPerslble in the extracting solvent (Warner et al 19R^
The Soxhlet technique is limited by the increased chemical "eacSvlJ; whLh'
can occur at the elevated temperature of the refluxing solvent the in?
creased extraction time which is required if the procedure is conducted
so^Tan^sedr6;: " *}' T^ ^ ^^ °* 'he blender -traction for
EJl and^edlment samples is that it works very well for dispersing particu-
983) A ^rraCtln? S°1Vent t0 Pr°m0te extraction efficiency (Warner et al.
«??',./ , Procedure uses a sonic probe to disperse the sample in an
extracting solvent. The sonification procedure is not as effective as the
blender for reducing the size of solid particles, although it is particularly
blIn£r8e 1983>- Adams (1983) compawd the efficiency of the
that the SoxhL3 J°S18J pr°Cedurs £or extracting aza-arenes. She observed
whne tL «nnif 5 procedures provided acceptable reproducibilities,
°C Ur! 83Ve P°°r reProd«cibilities for many compounds
P""dure which is ultimately selected for the Initial
extcton f na
rh»r I ? Jf S ° i 3u sedlment samPles will be selected according to the
tion should i" f the mf erlalS PreS6nt ±n the SafflPle- T116 bonier extrac-
tion should be adequate for the majority of samples; however, it may be
advantageous to use the Soxhlet extraction if a sample contains materials
which could be resistant to solvent extraction.
^ f ?! JJ0lVunt felected to extract organic compounds from soil is usually
restriction, ^n3?'"18^08 °f ^ ComP°unds P«sent. However, additional
J^M T I "? 7 t0 the 8electlon of solvents prior to biological
testing. Two main factors will influence the selection of a soil extraction
ZoinLE8^ Prl°V° blol°*lcal testin8- pi"t, the solvent must remove
both toxic and growth-stimulating substrates that could interfere with the
bioassays. These will include toxic compounds which could inhibit the
th^ ™ ""itagenic compounds and growth substrates, such as histidine,
that could mask the detection of weak mutagens. Brown et al. (1984b) reduced
164
-------
sss
.
sarnies fo/mutagenicity assays should also avoid grating arti vnts or
increase or decrease the tnutagenic potential of the soil extract. Solvents
their contaminants may alter the mutagenic potential of a ^st substance
during the extraction procedure through oxidation or through the formation of
radicals (Aeschbacher et al. 1983). Thus, the extraction and f"ct^n
procedure used to concentrate samples for mutagenicity testing should e
solvents and procedures that cannot chemically modify the test material.
A variety of solvents have been employed to extract organic compounds
from soil for chemical analysis. For the extraction of humic substances from
soil, Ogner and Schnitzer (1970) used benzene and ethyl acetate, while
Cifrulak (1969) used a benzene-methanol-acetone (2:1:1) -mixture. Several
Studies have used solvent extraction to determine the oil content of soil.
Jobson et al. (1974) used n-pentane, while Jensen (1975) used carbon tetra-
chloride, and McGill and Rowell (1980) used methylene chloride Methylene
chloride was also used by Wakeham (1979) to extract aza-arenes from sediment.
Carey and Gowen (1978) extracted polychlorinated biphenyls from soil with a
3-1 mixture of hexane:isopropanol. Each of these studies was concerned with
the efficient extraction of organic compounds from soil, and none addressed
the limitations associated with extractions for biological testing.
Only recently has research been conducted where soil or sediment samples
were extracted for analysis in a biological test system There have been two
publications reporting on the mutagenic activity of agricultural so±l'
Gozeelmann and Spitzauer (1982) used a 2:1 hexane: ace tone system to extract
Boll? DichLromethane was used by Brown et al. <1984a) ".extract three
agricultural soils for mutagenicity testing. A study examining the muta-
Snicity of sediment samples by Kinae et al. (1981) obtained two P rimary
fractions by successive extractions with diethylether and met hanol Ki nae et
al (1981) observed a higher level of mutagenicity in one of the ether ex
Sacts. although neithe/solvent gave consistently higher yields of hydro-
carbons Sato et al. (1983) also used ether to extract sediment samples.
However; prior to biological testing, Sato et al. (1983) further fractionated
thTIther extract using a series of solvents. Any one or a combination of an
assortment of solvents may be used to extract organic compounds from soil or
sediment samples. If the solvent will be evaporated prior to biological
testing, any solvent can be used, provided it does not generate artifacts and
does remove interfering substrates.
The USEPA (1982) recommends dichloromethane, and the results of Brown et
al (1984a) indicate that dichloromethane does not cause interference.
Although a 1:10 dichloromethane dimethyl sulfoxide solvent mixture does not
influence the mutagenicity of diagnostic mutagens in the Salmonella assay,
this solvent mixture can affect the results of other bioassays (Brown et al.
1984b). Thus, a more limited group of solvents can be used to dissolve the
165
-------
twth b1±?10glCal test±ng- <**«ie solvents which are com-
dichloromethane should be the primary solvent system used for extraction
' '
be investigated.
Fractlonatidn
*^''
group acid-base reactions (Adams et al. 1983). The liauid-HmHH
acid-base extraction method is based on the acidity constants fpKs) of
organic functional groups within the compound's molecular struc?u§e Com-
pounds which reside in the organic phase at low PH, low PK s?are Character-
ized as acidic. Compounds which reside in the organic phale at high pH are
characterized as basic, and compounds which exhibit no change in soluMlitJ at
an altered pH are neutral. Liquid-liquid extraction can be used to separate a
tinehe ,H Ura raCt°"S ? sele«
adjusting the pH of the aqueous phase. Brown et al. (1984b) employed this
SePate the dlchl°™*ethane extract of hazardous waste and
waste amend
Sato eTTl nq«^ f ° thrf Prlmary fractions ^c^. base and neutral).
' 1983)also used a ^quid-liquid acid-base extraction to
frct on o
tlxr £ f I the ether extract of sediment samples. A similar procedure is
and identif? M *S *' ^^ *°* ^^ 6t al« (1980> for 'he separation
et al (1980) u«°H ¥< ^^ Constltuents °f petroleum substitutes. Guerin
extractiof^n "Sed diethylether Jo obtain a primary extract with an acid-base
Tllll) n^J H J' ^"^ the Prlmary extract- Pelroy and Peterson
1981) obtained seven fractions of shale oil for mutagenicity testing using
tiSo°nCortheC°UP f ^ a 1^uld'li^id extraction. Foflowing'sol^ent'extrac8-
tion of the acid and base fractions, Pelroy and Peterson (1981) extracted the
^^"oxide to obtain neutral and polycyclic
bfactlons- Thus> -olvent extraction can^e used to
extract
«nd Sediment SamPles» and thls
extract
liquid acid-
Isolation
acid ^iT1 !eparatlon of the three primary fractions generated through
acid-base extraction may be required in some cases in order to isolate the
166
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mutagenlc components of a complex organic mixture. Chemical isolation pro-
cedures generally employ chromatographic techniques coupled with a separation
based on the polarity of the extracting solvents. The most simple technique
uses a silica gel column and is described by Warner (1976). Brown et al.
(1984b) employed this procedure to bioassay four neutral subtractions of
hazardous waste. Brown et al. (1984b) showed that subsequent elutions with
petroleum ether, followed by two elutions with dichloromethane:petroleum ether
(1:4) and a final elution with dichloromethane, yielded a saturate, aromatic
and two condensed ring fractions. Epler et al. (1978) used Florisil (reg-
istered trademark, Floridin Company) to separate the neutral fraction of crude
oil for biological testing. The procedures of Bell et al. (1969) were used to
elute fractions from the Florisil column with hexane,, hexane:benzene (8:1),
benzene:ether (4:1) and methanol. Sephadex LH-20 (Pharmacia Fine Chemicals)
gel has also been used to isolate bioactive components of a complex mixture.
Jones et al. (1977) describe a three-step fractionation procedure. The
mixture is first separated into lipophilic and hydrophilic fractions using
Sephadex LH-20 gel swollen with methanol/water and eluted with hexane. The
lipophilic fraction can be further separated into polymeric, sieved and hydro-
gen bonding constituents using a column of LH-20 swollen and eluted with
tetrahydrofuran. In the final step, the gel is swollen and eluted with
isopropanol to separate the sieved fraction into aliphatic, aromatic and
polyaromatic fractions. Toste et al. (1982) followed partition chromatography
using Sephadex LH-20 with high-performance liquid chromatography (HPLC) to
facilitate the organic and biological analyses of synfuels. Donahue et al.
(1978) also used HPLC to isolate mutagenic impurities in carcinogens and
noncarcinogens. A technique developed by Bjorseth et al. (1982) employed
thin-layer chromatography plates to separate the components of complex mix-
tures and to evaluate their mutagenic potential directly by means of the
Salmonella assay. Chromatographic techniques are useful for the separation of
the components of a complex mixture when chemical identification is required
or when the mutagenic constituents are present in dilute concentrations.
There are two primary disadvantages associated with generating a large number
of fractions using chromatographic techniques. First, the expense of con-
ducting multiple bioassays will be substantially increased when compared to
the cost for testing three to six primary fractions. In addition, extensive
separation of a complex mixture will make it difficulte to interpret the
interactions that will influence the toxicity of the mixture as a whole.
However, chromatographic fractionation may be necessary when toxic substrates
interfere with the bioassay or when mutagenic substrates are present in dilute
quantities.
All soil and sediment samples will require at least a preliminary extrac-
tion to obtain a sample for mutagenic characterization. However, extreme
caution should be exercised when interpreting the test results of a frac-
tionated mixture. In order to obtain the most accurate characterization, each
successive extract or fraction should be tested in at least one biological
test system. If the original extract is mutagenic, it should be separated
into acid, base and neutral fractions. Those fractions giving a positive
response in the bioassay will be further isolated using liquid/liquid separa-
tion and/or chromatographic techniques, followed by a retest in the bioassay.
167
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168
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REFERENCES
Abbondandolo A, Bonatti S, Corsi C et al. 1980. The use of organic solvents
in mutagenicity testing. Mutat. Res. 79:141-150.
Adams J. 1983. Adsorptivities of azaarenes on Flors^l. Dissertation for
Doctor of Philosophy in Chemistry, Texas A&M University. College Station, TX.
Adams J, Donnelly KC, Anderson DC. 1983. Hazardous waste streams. In: Brown
KW, Evans, GB, Jr., Frentrup BD, eds. Hazardous Waste Land Treatment.
Massachusetts: Butterworth Publishers, pp. 127-182.
Aeschbacher HU, Finot PA, Wolleb U. 1983. Interactions of hisitidine-
containing test substances and extraction methods with the Ames mutagenicity
test. Mutat Res. 113:103-116,
Ames BN, McCann J, Yamasaki E. 1975. Methods for detecting carcinogens and
mutagens with the Salmonella/mammalian microsome mutagenicity test. Mutat.
Res. 31:347-364.
Bell JH, Ireland S, Spears AW. 1969. Identification of aromatic ketones in
cigarette smoke condensate. Anal. Chem. 41(2):310-313.
Bjorseth A, Eidsa G, Gether J, Landmark L, Moller M. 1982. Detection of
mutagens in complex samples by the Salmonella assay applied directly on
thin-layer chromatography plates. Science. 215:87-89.
Brown KW, Donnelly KC, Thomas JC, Davol P, Scott BR. 1984a. Mutagenicity of
three agricultural soils. Sci Total Environ (in press).
Brown KW, Donnelly KC, Thomas JC. 1984b. The use of short-term bioassays to
evaluate the environmental impact of land treatment of hazardous industrial
waste. Draft final report. Grant No. R-807701-01. Washington, DC: U. S.
Environmental Protection Agency.
Carey AE, Gowen JA. 1978. PCB's in agricultural and urban soil. National
soils monitoring program. USEPA, Washington, DC. pp. 195-198.
Cifrulak SD. 1969. Spectroscopic evidence of phthalates in soil organic
matter. Soil Sci. 107(1):63-69.
Donahue EV, McCann J, Ames BN. 1978. Detection of mutagenic impurities in
carcinogens and noncarcinogens by high-pressure liquid chromatography and the
Salmonella/microsome. Cancer Res. 38:431-438.
169
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5: *- of
Kinae N, Hashizume T, Maki.ta T et al. 1981. Studies on the toxicitv of
-1ii1Sf1'IE2rIi..!hSfSS!y 0£ the sedl"ent --- --
irfE?" ^VMande< M«" a1' 1M1- A° e"al««lon of tests using DKA-
f
of oil content of
170
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Ogner G, Schnitzer M. 1970. The occurrence of alkanes in fulvic acid, a soil
humic fraction. Geochimicaet Cosmochimica Acta. 34:921-928.
Pelroy RA, Peterson MR. 1981. Mutagenic characterization of synthetic fuel
materials by the Ames/Salmonella assay system. Mutat. Res. 90:309320.
Sato T, Momma T, Ose Y, Ishikawa T, Kato K. 1983. Mutagenicity of Nagara
River sediment. Mutat. Res. 118:257-267.
Scott BR, Dorn GL, Kafer E, Stafford R. 1982. Aspergillus nidulans; systems
and results of tests for induction of mitotic segregation and mutation II.
Haploid assay systems and overall response of all systems. A report of the
USEPA's Gene-Tox Program. Mutat. Res. 98:49-94.
Suzuki J, Sakamasu T, Suzuki S. 1982. Mutagenic activity or organic matter
in an urban river sediment. Environ. Pollut. Ser. A. 29:91-99.
Toste AP, Sklarew DS, Pelroy RA. 1982. Partition chrpmatography-high-
performance liquid chromatography facilitates the organic analysis and
biotesting of synfuels. J, Chromat. 249:267-282.
USEPA. 1982. U. S. Environmental Protection Agency. Office of Water and
Waste Management. Test methods for evaluating solid waste: physical/chemical
methods. 2nd ed. Washington, DC: U. S. Environmental Protection Agency.
SW-846.
Wakeham SG. 1979. Azaarenes in recent lake sediments. Environ. Sci.
Technol. 13(9):1118-1123.
Warner JS. 1976. Determination of aliphatic and aromatic hydrocarbons in
marine organisms. Anal. Chetn. 48(3):578-584.
Warner JS, Landes MC, Slivon LE. 1983, Development of a solvent extraction
method for determining semi-volatile organic compounds in solid wastes. In:
Conway RA, Gulledge WP, eds. Hazardous and Industrial Solid Waste Testing:
Second Symposium, ASTM STP805. American Society for Testing and Materials,
pp. 203-213.
Withrow WA. 1982. Mutagenicity of roadside soils. Thesis for Master of
Science in Environmental Engineering, Illinois Institute of Technology.
Chicago, IL.
171
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SOILS AND SEDIMENTS WORKGROUP REPORT
Workgroup Tasks
Summary of Tasks —
°f
; riiziri^ i-sr- (usln8
5. Storage of extract.
6. Method of extraction.
7. Restriction of assay.
8' extract Sami>le eXtraCtl°n ^"^ng initial Ames test on crude
9. Need for replicate samples and assay.
1U. Sample handling to prepare.
11. Proper negative control.
Key Peer Review Comments--
There are no unresolved peer review comments.
Summary of Workgroup Progress-
Major questions considered and rationale for choice:
on tS ^/JM™";,*0 Ch°°Se different extraction solvents
ossil soil contaminants, this information will not
PJ°Cedure should us« the Soxhlet method routinely,
pr°Cedure is ^H-known and widely used. However other
'
instigator shouU have the fedo.
172
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Summary of Key Workgroup Discussions
Major Consensus Opinions—
There was uniform agreement that labs should use spiked samples to demon-
strate their ability to extract organics from soil. We listed several methods
that could be used to determine efficiency of extraction.
We also did not wish to restrict the principal investigator to one
extraction method; thus, we listed the Soxhlet method as the primary one, but
gave an alternative method that ultimately may be faster. Similarly, the
procedure to further fractionate the extract following the initial Ames test
was left open. We listed the most widely used method? acid/base/neutral
techniques, and then a second method, HPLC. This HPLC method may ultimately
be the most useful for the future, and we felt it was important to have it
listed as an alternative procedure to be used at the discretion of the re-
searcher. The question of internal standards or a universal soil blank for
negative controls was asked in the general meeting. We concluded that the use-
of an internal standard for a sample to be tested in a bioassay is inappro-
priate, since it would obscure the analysis of the sample itself. A universal
soil blank is not needed as a negative control; proper characterization of
the solvent negative control is sufficient as a "blank."
Other Data or Information Requirements
An important information gap that we found in developing this protocol
was quantitative data on the stability of organic pollutants in soil under
various storage conditions. To bridge this gap, a research program needs to
be initiated to measure the degradation of a variety of compounds spiked into
soil under various storage conditions. The effects of storage temperature
(-4 C to -80 C), soil moisture, soil type and storage time (1, 3, 6, 9 and
12 months) should be determined as a minimum. Another problem that could be
addressed in this study is the stability of the compounds in autoclaved soil.
That work is important because totally stable soil performance evaluation
samples are not presently available.
Another information gap is in the area of solvent extraction of mutagens
from soil. The recoveries of a variety of organic compounds of different
polarities and/or functional groups should be extracted from several soil
types adjusted to pH 4, 5, 7 and 8 with methylene chloride only, methylene
chloride followed by methanol, a combination of methylene chloride and
methanol (9:1 v/y) or other solvent mixtures or sequences suitable for ex-
traction of soils/sediment. The recoveries of the compounds and the suita-
bility of the different solvent systems for compounds of different polarity
should be determined. In addition, the effect of soil pH on the extraction of
various compounds should be quantitated and optimized.
A final area of research that needs to be investigated is the fractiona-
tion of crude extracts. The effect of acid and base fractionation on a
variety of mutagenic extracts could be compared side by side with the HPLC
fractionation technique discussed by Dr. Wilson Tabor at this meeting. The
sum of the (revertants-baekground)/gram soil for each of the techniques could
173
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174
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PROTOCOL FOR THE PREPARATION OF SOIL AND SEDIMENT SAMPLES FOR MUTAGENICITY
TESTING
1.0 Scope and Application
1.1 Application— *
The following protocol has been developed for the preparation of soil
and sediment samples for mutagenicity testing. This protocol is designed to
provide samples which accurately represent the mutagenic potential of the soil
or sediment sample initially extracted and, if necessary, sufficiently
fractionate the original material to Isolate bioactive materials. Fractiona-
tion should only be required if the toxicity of the crude extract prevents
determination of the mutagenic potential. Included in this protocol are
procedures for sample preparation, extraction and fractionation. The use of
standardized procedures for mutagenicity testing will increase the accuracy of
the bioassay results and allow interlaboratory comparisons to be made.
1.2 Method Detection Limits—
The method detection limit will be specific for each compound and each
biological test procedure. In addition, the interactions of the components of
a complex mixture will also affect the detection limits of a bioassay. Thus,
sensitivity should be determined for each experiment using solvent and
positive controls.
1.3 Limitations—
This protocol includes procedures for preparing solvent-extractable
organic compounds for mutagenicity testing. The protocol does not include
procedures for the preparation of inorganic (i.e., heavy metals) or volatile
organic constituents, which will be removed or isolated in the preparation of
the solvent-extractable organic constituents.
2.0 Summary of Method
This protocol describes primary and optional procedures for the
isolation and fractionation of soil and sediment samples for mutagenicity
testing. The protocol includes a Soxhlet, blender and Bonification procedure
for solvent extraction of organic compounds. In addition, two procedures,
a liquid-liquid, acid-base extraction and an HPLC extraction, are provided for
the fractionation of the solvent-extractable organic compounds.
3.0 Definitions
a. Soil - The unconsolidated material on the earth's surface that is
less than 2 mm in diameter and capable of supporting plant growth.
175
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b. Sediment - Soil material deposited and remaining in an aquatic
environment.
c. Fractionation - The separation of solvent-soluble compounds from a
bulk matrix.
d. Extraction - Manipulation of chemical and/or physical conditions
within a sample matrix which effectively isolates and enriches a
specific class of compounds sensitive to the manipulation.
4.0 Interferences
r ^
Two types of interferences can be anticipated in a mutagenicitv assav
Interference due to growth substrates (e.g., hisMdine) should 1e removed *'
using the extraction procedure (Sections 7-9), while interference caused by
toxic compounds may be reduced by fractionation procedures (Sections 10 and •
5.0 Safety
This procedure is to be used for the collection and preparation of
soil or sediment samples for mutagenicity testing. All samples that are
collected should be treated as biohazardous material. Thus! appropriate
safety precautions must be taken.
a.
Samples from an area where a spill of a known or suspected high-
level mutagen has occurred should be taken by properly trained
personnel wearing disposable full-coverage suits and masks.
Bareis et al. (1983) suggest that in areas where the anticipated
contaminant levels are more than 5 ppm above background, indi-
viduals should also wear a self-contained breathing apparatus.
These precautions will not be necessary in gathering routine
samples from areas when no release of mutagens has occurred.
The threads of the sample collection bottles should be wiped free
of any residue before sealing the jar. After the jar is sealed,
it should be wiped clean and enclosed in a plastic bag before
transport to the lab or storage facility. Materials used to wipe
the jars should be disposed properly.
Sample bottles should be removed from the plastic bags in a high
draft hood and examined to determine that they are not cracked or
broken.
d. All manipulations involved in the extraction or fractionation of
samples should be performed in a high draft hood.
The solvent exchange of extracts and fractions into DMSO and the
manipulation of the DMSO solutions must be handled in a high draft
hood. In addition, the DMSO solutions must be handled with two
176
c.
e.
-------
pairs of gloves. Special precautions are necessary with these
potentially mutagenic samples because of the unique biological
transport of DMSO.
f. Spiking of soil samples with mutagenic compounds that serve as
positive controls is to be done in a hood with great care. Two
pairs of gloves should be worn, and the dissolved mutagens should
be applied to the soil at close range in order to minimize drift.
g. All samples, extracts or fractions must be handled with caution
and, until proven otherwise, they should be treated as mutagens.
h. All contaminated material should be handled and disposed following
proper procedures for the disposal of hazardous waste.
6.0 Sample Collection, Preservation and Handling
Collection, preservation and handling of soil and sediment samples for
biological testing must be performed in such a manner that mutagenic activity
is neither lost nor generated. It is especially important that samples of
soil or sediment collected for mutagenicity testing provide an accurate
representation of the sampling location. Sampling procedures are discussed in
Peterson and Calvin (1965) and deVera et al. (1980). The USEPA (1980) recom-
mends a one-quart, wide-mouth, screw-cap, glass bottle with a Teflon lid liner
for storing soil and sediment samples. At the time of collection, the bottle
should be filled nearly to the top with the soil or sediment sample. If the
sample is collected from below water, the threads and sealing surfaces should
be washed off with the sample water. Sediment samples should be topped off
with sample water and sealed with the Teflon-lined screw cap. Soil and
sediment samples should not be air or oven dried before storing at 4 C.
Samples stored more than 48 h should be stored at -10 C. Care should be taken
with anaerobic samples to avoid contact with oxygen. A sufficient sample
should be collected to provide adequate residue for the needs of the bioassay
and a reserve sample. In most cases, approximately 500 g should be adequate
for a soil contaminated; by oil or .other organic materials. For uncontaminated
soils or soils with a high moisture content, 2 to 3 kg of sample may be
required.
Soil and sediment samples to be analyzed for mutagenic potential
should be labeled by procedures prescribed for handling hazardous waste.
Primary particles greater than 2 mm in diameter must be removed by sieving as
a first step in sample preparation. It may be necessary to break aggregates
by dicing or crushing to facilitate subsampling. Samples must be homogenized
and properly subsampled to assure that the results are not biased by spot
concentrations in the soil. This can best be done by repeatedly quartering
the samples to avoid the biased selection of a given particle size. Quarter-
ing is done by spreading the sample on a clean piece of paper (typically 60 cm
by 60 cm). The centers of opposite edges of the material are then raised,
separating the sample in half. The centers of the other two opposite edges
are then raised, dividing the sample into quarters. Three quarters of the
sample is returned to the sample container, and the remaining quarter is
requartered until the desired sample size is achieved. A duplicate sample
177
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rpr large anounts °f
separated from the soilTy filtration of th eXtra"io11: The »«« can be
or by centrifuge. The weLht of l*T, fi TP ! Under " ni«°8^ Mantel
and after the wakr is reeved If thf r^" S?°"ld *e deter»i»^ before
should be prepared a.ord^lo tnf '
7'° Extraction Protocol (Soxhlet)
a.
7.1 Apparatus and Equipment—
Soxhlet ^tractor - 40-nnn ID, with 500-mL round-bottom flask.
Kuderna-Danish (K-D) apparatus with a three-ball Snyder column
UUm r eVap°rator with ch"^d solvent receiving
mateAOl., ' -» ' aPP"^-
mately 400 mm long, wxth a coarse fritted plate at the bottom and
an appropriate packing medium. This is used as a drying co?umn?
7.2 Reagents and Consumable Materials __
•a. An appropriate solvent or solvent mixture of HPLC grade. In most
«
7.3 Procedure —
3' sulfate'and n? °J ^r^ Sample Wlth 10 8 °f ^ydrous sodium
with a 2faf/rod V § " ^tractlon thlmb1^ Mix thoroughly
with a glass rod. If any problems are encountered, e.g., the
sample clogs, the thimble, an alternative is to place a iiuTof
ci± W°°1;,lrVthe extraction chamber, transfer ?he sample Ltl the
chamber and then cover with another plug of glass wool!
b. Place thimble and glass rod in the Soxhlet extractor Assemble
rt0F in* flasks -ntaining 300 mL of s'lve" "nd a
(solvent-extracted). Apply heat to the boiling
178
-------
flask and adjust the temperature to give six flushings per hour
(caution: avoid excess temperature). Extract the sample for
16 h.
c After the extraction is complete, the solvent extract should be
cooled and then filtered and dried by passing it through a 10-cm
column of solvent-washed sodium sulfate.
d The procedure used for solvent reduction will be the same for all
extraction procedures and is described at the end of this
protocol.
\
8.0 Extraction Protocol (Blender^
8.1 Apparatus and Equipment —
a. Explosion-proof blender, accommodating stainless steel or boro-
silicate glass container with 1.2-L capacity.
b Kuderna-Danish apparatus with a three-ball Snyder column or vacuum
rotary evaporator with a chilled solvent receiving flask.
Chromatography column - Borosilicate glass, 20-mm ID, approxi-
mately 400 mm long, with a coarse fritted plate at the bottom and
an appropriate packing medium. This is used as a drymg column.
8 ? Reaeents and Consumable Materials —
a In appropriate solvent or solvent mixture of HPLC grade. In most
instances, dichloromethane can be used; subsequent extraction with
methanol may be necessary to recover polar compounds.
b. Anhydrous sodium sulfate, ACS (purified by heating at 400 C for
4 h in a shallow tray).
c.
g of soil or sediment sample with 25 g of anhydrous sodium
sulfate and place in blender jar.
b. Add approximately six volumes (150 mL) of the extracting solvent
to the blender jar and screw cover down tightly.
c Blend mixture for 30 sec in a fume hood. Decant solvent and
repeat extraction twice with 75 mL of fresh solvent or until
extracting solvent remains colorless.
d. Combine solvent extracts and then filter and dry by passing
through a 10-cm column of solvent-washed sodium sulfate.
e. The procedure used for solvent reduction will be the same for all
extraction procedures and is described at the end of this
protocol.
179
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9'° Extraction Protocol ^Bonification)
9.1 Apparatus and Equipment—
*
9.2 Reagents and Consumable Materials—
a. An appropriate solvent or solvent mixture of HPLC erade Tn ™ *
instances, dichloromethane can be used; subsequent'ex^ctit^th
to recover polar compounds.
b.
9.3 Procedure—
a.
50 g of
b. Add approximately six volumes (150 mL) of the extracting
ana agitate with a "OS u 5«r,-j«^*-__ ..J^L , „ . . . 6
i , . . • "• **• <~ui ".x i • ^j—cm proD
L cm below the surface of the solvent for 3 min on a
duty cycle with a power input of 50% full scale.
c. Decant the solvent and remix the soil or sediment sample.
d' ^Tfhhe extract±on twlce with 150-mL volumes of solvent or
until the extracting solvent remains colorless.
e. Combine solvent extracts and then filter and dry by passing
through a 10-cm column of solvent-washed sodiumsulfate.
f' extracting ""? f°r ^^ redu«ion will be the same for all
protocol Pr°CedureS and ls described at the end of this
10'° Solvent Reduction Procedure
a.
rtas- technl«ues ^y be used, Adams
with th ? ^tained the best recovery using a rotary evaporator
with the solvent-receiving flask immersed in an ice bath Thus
bTSSSL f°rThb0th ? ^ 3nd r°tary -aporator concentration "ill
be provided The solvent extract should be collected in a 500-mL
K-D flask fitted with a 90-mL graduated concentrator tube The
IoSrtoCt?25fmLaSofathd ^^ ^^ C°
xuu to 125 mL of the extracting solvent.
180
-------
Concentration using a K-D apparatus is accomplished by adding one
to two solvent-treated boiling chips to the flask and attaching a
three-ball Snyder column. Prewet the Snyder column by adding
about 1 mL of solvent to the top. Place the K-D apparatus on a
steam or hot water bath so that the concentrator tube and the
entire lower rounded surface of the flask is bathed in hot water
or vapor. Adjust the vertical position of the equipment and the
water temperature to complete the extraction in 15 to 20 min
(approximately 45 C). At the proper rate of distillation, the
balls of the column will actively chatter, but the chamber will
not flood. When the volume of the liquid is less than 10 mL,
remove the K-D apparatus and allow to drai^n for at least 10 min
while cooling.
If a rotary evaporator is to be used, transfer the solvent extract
to a 500-mL rotary evaporator flask. The extractor flask and
sodium sulfate column should be washed with 100 to 125 mL of the
extracting solvent. Adjust evaporator flask water bath tempera-
ture to 45 C, circulate chilled coolant through condenser, attach
vacuum port to aspirator or other vacuum source and place solvent-
receiving flask in an ice bath. Reduce solvent volume to less
than 20 mL on rotary evaporator and transfer with rinsing to a
sample collection tube.
An aliquot of the solvent extract may be removed for gravimetric
analysis of solvent-extractable organic carbon or for fractiona-
tion prior to the final concentration step. The final concentra-
tion step will employ a solvent exchange using DMSO. The volume
of the initial solvent extract should first be carefully reduced
to 1 mL at 40 C under a gentle stream of nitrogen. Because the
solvent evaporates rapidly, it is important that this operation be
done under constant surveillance to ensure that the volume is not
reduced below 1 mL. It is also necessary to warm the samples
slightly, either by hand or water bath at <40 C, to prevent
condensation of atmospheric moisture in the sample by evaporative
cooling.
Add 1 mL of DMSO to the sample and mix by gentle agitation. The
total sample volume is reduced under a stream of nitrogen to
1.5 mL. Another 1 mL of DMSO is added and mixed, and the volume
is reduced to 2.25 mL. The exchange is repeated with another 1 mL
of DMSO, and the volume is reduced to a final volume of 3 mL.
Other volumes of DMSO may be used if 3 mL of DMSO does not give a
suitable sample concentration.
11.0 Fractionation Protocol
The least complex and most widely accepted protocol for fractionation
of an organic extract employs a liquid-liquid extraction to partition acid,
base and neutral constituents (Figure 18). This procedure is recommended for
evaluating solid waste by the USEPA (1982) and Adams et al. (1983) and was
used by Brown et al. (1984b) to isolate mutagenic fractions from waste-amended
181
-------
Crude Sample in MeCl,
Extract with
IN HC1
Organic
Acid and Neutral
Extract
Organic
* I Neutral j
with IN NaOH
Aqueous
1HC1
Extract
Organic
with MeCl,
Aqueous
ppt.
Base
Extract
Organic
with MeCl,
ppt.
I Base I
Water Soluble
Aqueous
Water Soluble
Figure 18. Fractionation scheme for soil and sediment samples.
-------
soil. An optional procedure which employs HPLC to fractionate samples is also
included for samples where it is desired to avoid the risk of hydrolyzing
mutagenic compounds with NaOH.
11.1 Apparatus and Equipment—
a. Separatory funnel with a Teflon stopcock (2,000 mL).
b. Kuderna-Danish apparatus with a three-ball Snyder column or vacuum
rotary evaporator with a chilled solvent receiving flask.
c. Chromatography column - Borosilicate glass, 20-mm ID, approxi-
mately 400 mm long, with a coarse fritted plate at the bottom and
an appropriate packing medium.
d. Evaporator flasks as appropriate for collecting fractions.
11.2 Reagents and Consumable Materials—
a. Sodium hydroxide - (ACS) IN in distilled water (chilled to 4 C).
b. Hydrochloric acid - (ACS) IN prepared by mixing concentrated HC1
and distilled water (chilled to 4 C).
c. An appropriate solvent or solvent mixture of HPLC grade. In most
instances, dichloromethane can be used.
d. Anhydrous sodium sulfate - (ACS) purified by heating at 400 C for
4 h in a shallow tray.
e. Distilled water - chilled to 4 C.
f. pH test paper providing readings over entire pH range from 0-14.
11.3 Procedure—
a. Dissolve 10 g of the extract in 30 mL of solvent. In most
instances, the solvent will be dichloromethane.
b. Place sample in a separatory funnel.
c. Adjust the pH to 1-2 with sulfuric acid (approximately 100 mL).
Partition the sample into the two phases by shaking the funnel for
1 min with periodic venting to release pressure (point funnel away
from the analyst).
d. Allow the fractions to separate in an upright stand (10-min
minimum). If the emulsion interface between layers is more than
one-third the size of the solid layer, the analyst must employ a
mechanical device to complete the phase separation. The optimum
mechanical technique depends on the sample, but may include
stirring, filtration of the emulsion through glass wool or cen-
trifugation.
183
-------
contain the Jf "i" *' the 1OW6r phaSe' Thls Phas* "ill
contain the acid and neutral fractions. Collect the upper phase
in a flask and label. The upper phase contains the base fraction.
f . Reextract the solvent layer twice more with IN HC1 at pH 1-2
(approximately 50 mL each). Cotnbine the aqueous base extracts and
m.
n.
layer obtalned ln StePs e and f ^ PH
1N NaOH to the base fractlon
h. Allow the fractions to separate in an upright stand (10-min
te
Blower
and label. Add 100 mL of dichloromethane and repeat extrac
P "0
t2-13tbyhthfadditionSorVent ^^ °btaltled in SteP8 e and f to pH
Partition the sample into two phases by shaking the funnel for
from^he'ana^tK0 Ventln8 *" releaS6 Pr6SSUre (P°lnt funnel aw^
stand
v ^-sswrs s
mechanical device to complete the phase separation.
con/I89^0 l3yer T111 bS the lwer Phase- Thls Phase will
anS Ube ?hT ^ ^^ Colle" the upper Jhase in a flask
and label. The upper phase contains the acid fraction.
r!^M r°1VJfnt layer tWlCe more wlth 1N NaOH (approximately
each). Combine the aqueous acid extracts.
The organic layer will contain the neutral fraction.
o. Adjust the PH of the aqueous layer obtained in Steps 1 and . to PH
200 mL 0^e,ad.d,ition °f 1N HC1 -d extract the acid '
Allow the fractions to separate in an upright stand (10-min
ont'third th the 6f ^ ^^ be?W6en la^ers is -- than
mechanical dL>2% the1S°lvent layer, the analyst must employ a
mectianical device to complete phase separation.
184
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q. The organic layer will be the lower phase. This phase will
contain the acid fraction in dichloromethane. Collect the lower
phase in a flask and label. Add 100 mL of dichloromethane and
repeat the separation to collect a second acid fraction in
dichloromethane, which is added to the first.
r. The dichloromethane fractions obtained in Step i (base fraction),
Step n (neutral fraction) and Step q (acid fraction) are concen-
trated separately using the procedures described in Section 10.
The corresponding aqueous fractions and any precipitate remaining
represent additional fractions which, in most instances, will not
be tested in the bioassay. v
11.4 Fractionation Procedure (HPLC)—
An alternate procedure which can be used to fractionate the solvent
extract of soil and sediment samples employs an isocratic gradient on an HPLC.
This procedure is less likely to hydrolyze organic compound than the acid-base
extraction and is described in detail in the section on Drinking Water.
12.0 Calibration
Due to the variety of matrix conditions exhibited by soil and sediment
samples, verification of the solvent extraction efficiency must be conducted
on each batch of soil or sediment samples. The procedure outlined below is
designed to meet the above objective:
a. Select 25 g of sample typical of the lot to be assayed, as
described above. Cool the sample and store in a dry place.
b. Prepare a solution of 250 mg/mL 2-nitrofluorene and 2-amino-
anthracene in methylene chloride.
c. Spike the matrix with 1 mL of spike solution and allow to stand
for 24 h. Extract the matrix according to the procedure in
Section 7. Quantify the extract for the spike compounds.
Alternatively, extraction recoveries can be measured by another
suitable technique that has been validated. Two possibilities
are (1) determination of the amount of deuterated pr±o^ty
pollutant surrogates by GC/MS or (2) determination of C-labeled
mutagens by scintillation methods. A recovery of 80% of each
spike compound is considered acceptable. Below 80% recovery
indicates a potential problem. For some difficult cases, 70% may
be acceptable; however, 70% is not acceptable on a routine basis.
Listed below are several suggestions for examination when the extrac-
tion efficiency is below specification:
a. Recalibrate the HPLC and rerun the analysis.
b. Reexamine dilutions, weighing, etc.
c. Start from the beginning with the same matrix.
185
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d. Start from the beginning, but choose a different sample from the
same series.
e. Start from the beginning with a matrix from a different source.
Once the presence and quantity of the spikes have been established',
the extract can be used to verify the detection limit of the chosen mututation
assay. Note; Storage of this extract is subject to the same restrictions as
the sample extracts. Standard samples of mutagens should be stored for no
longer than these solutions have been demonstrated to be stable.
13.0 Quality Control
All laboratories preparing or testing soil and sediment samples should
follow a Quality Assurance/Quality Control program. The Quality Assurance
program ensures that all laboratory or field procedures are performed in a
scientifically sound manner. The Quality Assurance program should be based on
the guidelines set forth in "Good Laboratory Practice Regulations" (FDA 1979) '
and "Handbook for Analytical Control in Water and Wastewater Laboratories"
(USEPA 1979). As a minimum, the Quality Assurance program should address the
following points:
a. Obtain sample, fill in the required information on a chain-of-
custody form. Of critical importance are recording pH of sample
as soon as possible (on-site, if practical) and storage in an
amber glass bottle with a Teflon-lined cap at 4 C or lower.
b. Maintain sample usage form. Record total amount of sample, where
stored and amounts sent to testing laboratories. Before subsample
is removed for extraction, the soil must be homogenized to ensure
reproducibility of duplicate extracts. Record in raw data when
sample was homogenized, date and technician.
c. Dry sample. Record conditions and time needed and amount of
Na SO, used.
d. Perform extraction. Record amount of material, volume of solvent,
lot number, supplier, grade of solvent and duration of extraction.
e. Reduce solvent volume and add DMSO. Record amount of DMSO added
and final volume of extract. Measure total organic material, if
desired.
f. Solvent control for bioassay. Reduce an equivalent amount of
solvent and add the same amount of DMSO as needed for the extract.
Use equal volumes of negative solvent control in bioassay.
g. Determinations of solvent control and samples spiked with positive
controls will be performed as QC checks with each independent
bioassay run. The results of those determinations will be
recorded in a log book.
186
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h.
i.
SOPs for techniques used in the laboratory will be written, and
copies of those'documents will be kept with the QC log book.
As a minimum, one of every ten extractions will be conducted in
triplicate to verify the reproducibility of the method.
of each laboratory study.
14.0 Calculations
Total Weight = TW - (soil weight + water weight)
Soil Weight = SW - (TW-WW)
Water Weight = WW = (TW-SW)
Moisture Content = %W - ^^SL x 100%
TW
Dry Weight of Sample = D = (TW-(SW-%W))
D gram equivalents
Extract is expressed as: DMSO ' ^milter
15 o Precision and Accuracy
"
s
..
187
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REFERENCES
Do^n/'f H8i' A5|s°rPtlvit±es of azaarenes on Florisil. Dissertation for
Doctor of Philosophy in Chemistry, Texas A&M University, College Station, TX.
Adams J, Atlas EL, Giam CS. 1982. Ultratrace determination of vapor-phase
nitrogen heterocyclic bases in ambient air. Anal. Chem. 54(9) : 1515-1519
Br ™ Drnelly™C' Anderson DC« 1983- Hazardous waste streams. In:
M~r h ' ?rnS'* ' Jr" FrentruP BD> eds- Hazardous Waste Land Treatment
Massachusetts: Butterworth Publishers, pp. 127-182.
Bareis DL, Cook LR, Parks GE. 1983. Safety plan for construction of remedial
action, in: National Conference on Management of Uncontrolled Hazardous
SSrtai^n^rS" 31 Z ?°Vember 2» 1983' Silver Spring, MD: Hazardous
Materials Control Research Institute.
Brown KW, Deuel LE, Thomas JC. 1982. Soil disposal of API pit wastes
Final Report to USEPA. Grant No. R805474013. wastes.
Brown KW Donnelly KG Thomas JC, Davol P, Scott BR. 1984a. Mutagenicity of
three agricultural soils. Sci. Total Environ, (in press).
Brown KW, Donnelly KC, Thomas JC. 1984b. The use of short-term bioassays
to evaluate the environmental impact of land treatment of hazardous industrial
mStlll P r ^eP°f' °rant N0> R-807701-01- Washington, DC: U. S. Environ-
mental Protection Agency.
deVera ER, Simmons BP, Stephens RD, Storm DD. 1980. Samplers and sampling
procedures for hazardous waste streams. EPA-600/2-80-018.
Donahue EV, McCann J, Ames BN. 1978. Detection of mutagenic impurities in
carcinogens and noncarcinogens by high-pressure liquid chromatography and the
Salmonella/microsome. Cancer Res. 38:431-438.
Donnelly KG, Brown KW. 1981. Development of laboratory and field studies to
determine the fate of mutagenic compounds from land applied hazardous waste.
22423C Land Dlsposal: Hazardous Waste. EPA-600/981-002b.
FDA. 1979. Food and Drug Administration. Good laboratory practice regula-
pp?ni-219,aAjrnC1975Cal lab°rat°ry Stm!y' Federal ^gister Vol. 21, No. 58,
188
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Goggeliaann W, Spitzauer P. 1982. Mutagenic activity, content of polycyclic
aromatic hydrocarbons (PAH) and humus in agricultural soils. Mutat. Res.
89:189-190.
Guerin MR, Ho CH, Rao TK, Clark BR, Epler JL. 1980. Separation and
identification of mutagenic constituents of petroleum substitutes. Intern.
J. Environ. Anal. Chem. 8:217-225.
Jones AR, Guerin MR, Clark BR. 1977. Preparative-scale liquid chromato-
graphic fractionation of crude oils derived from coal and shale. Anal. Chem.
49(12):1766-1771.
Peterson RG, Calvin LD. 1965. Sampling. Chapters/ In: Black LA et al.,
eds. Methods of Soil Analysis, Part I. Madison, WI: Agronomy Society of
America, pp. 54-72.
Toste AP, Sklarew DS, Pelroy RA. 1982. Partition chromatography-high-
performance liquid chromatography facilitates the organic analysis and
biotesting of synfuels. J. Chromatogr. 249:267-282.
USEPA. 1979. U. S. Environmental Protection Agency. Handbook for
analytical control in water and wastewater laboratories. EPA-600/4-79-019.
USEPA. 1980. U. S. Environmental Protection Agency. Environmental
Monitoring and Support Laboratory. Interim methods for sampling and
analysis of priority pollutants in sediments and fish tissue. Dralt.
Cincinnati, Ohio: U. S. Environmental Protection Agency.
USEPA. 1982. U. S. Environmental Protection Agency. Office of Water and
Waste Management. Test methods for evaluating solid waste: physical/chemical
methods. 2nd ed. Washington, DC: U. S. Environmental Protection Agency.
SW-846.
Warner JS. 1976. Determination of aliphatic and aromatic hydrocarbons in
marine organisms. Anal. Chem. 48(3):578-584.
Warner JS, Landes MC, Silvon LE. 1983. Development of a solvent extraction
method for determining semivolatile organic compounds in solid wastes. In:
Conway RA, Gulledge WP, eds. Hazardous and Industrial Solid Waste Testxng:
Second Symposium, ASTM 805, pp. 203-213.
Withrow WA. 1982. Mutagenicity of roadside soils. Thesis for Master of
Science in Environmental Engineering, Illinois Institute of Technology.
Chicago, IL.
189
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SECTION 7
WASTE SOLIDS
REVIEW OF THE LITERATURE
Introduction
Complex mixtures may be composed of a diverse mixture of organic and
inorganic compounds present in a variety of matrices (i.e., solids, liquids •
and gases) They can be obtained from such widely different sources as the
addrlssesnther ^n^turln« and treatment processes. The following document
genicity testing. * C°^ ** mixtures from a solld matrix for muta-
Solid Matrix - Definition—
A solid is defined as having relative firmness, coherence of particles or
50™^°°?*.° ^?T aS mat^r that 1S n0t Uquld Ot 8aseous- ^ the present
document, it will be considered to include tarry materials that are sticky or
viscous, such as coal tar sludge and adhesives wastes. It will, however,
exclude soil and sediments or solid particulates suspended in a gaseous
medium, since these are discussed elsewhere.
The Reason Separation is Necessary—
Many of the complex mixtures are highly toxic to the bioassays. This
toxicity in many instances overwhelms the detection of mutagenicity However
Qhna?5\° *Pi°88l?le interactions between components of a complex mixture, care
should be taken in interpreting the biological results using fractionated
complex mixtures.
Criteria for Sample Preparation—
Various guidelines can be suggested for choosing a sample preparation
scheme for use in bioassay evaluation;
a. The selected method should provide a sample that is representative
of the original solid matrix sampled within normal limits of
biological accuracy and precision.
b. It should not introduce artifacts or change or remove constituents
from the complex mixture.
c. The selected methods should be sufficiently tested to have
established their validity.
190
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d The procedure should be simple and cost effective. Methods should
' be sufficiently rapid to permit repetitive use in the examination of
large numbers of samples,
e As far as possible, the method should minimize the exposure of
workers to potential hazards from both the sample itself and the
procedure.
f. The methodologies chosen should be compatible with standard
analytical techniques.
i The procedure should remove toxic or growth-stimulating substances
which may interfere with the bioassay without altering the overall
composition of the sample.
Sources and Sample Collection
The first critical step in preparing a sample for biological analysis is
to collect a representative portion of the material to be evaluated. To
ensure that the analysis does not over- or underestimate the potential geno-
toxic effect, representative samples must be obtained. A representative
sample contains all constituents in the same ratio present in the bulk
material sampled. The probability of achieving this goal is enhanced by
compositing multiple samples. These samples can be composited prior to
subsamplinl for extraction or other procedures (USEPA 1982). In some in-
stances heating or sampling the bulk material at elevated temperatures allows
a more homogeneous sample to be collected. However, discretion should be used
in utilizing high temperatures, as loss of volatile material may occur. In
all instances, mixing should precede sampling where feasible .
The diverse sources of complex solid mixtures and particulates
necessitate using a number of different samplers. Table 6 summarizes the
recommended samplers for particulates of various sizes and sources of origin
(see de Vera et al. 1980).
It is very important that all these samplers be thoroughly cleaned and
free from contamination both prior to and between samplings. One simple
method to accomplish this is to wash with the solvent to be used. Storage
containers should be similarly cleaned. Glass or Teflon should be used as
sample containers. The reason for this is that plastic containers are often
coated with plasticizers that interfere with biological analysis as well as
organic analysis (USEPA 1982).
Extraction
Physicalj:reatment--^^ ^ efficietlt extraction of a solid matrix, it may be
necessary to pretreat the material prior to chemical extraction. If the
sample:
Contains particles of too large a size - Pretreat the sample by
grinding. The necessity for grinding and the choice of apparatus
a.
191
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Waste type
——- —
Slurries
Dry solids
Sticky or moist solids
and sludges
TABLE 6. SAMPLERS RECOMMENDED FOR VARIOUS TYPES OF
SOLID WASTE (USEPA 1982)
Waste location or
container
Sampling apparatus
Tanks, bins, pits, ponds, Weighted bottle, dipper
lagoons FF
Drums, sacks, piles,
trucks, tanks, pits,
ponds, lagoons
Drums, tanks, trucks,
sacks, piles, pits,
ponds, lagoons
Thief, scoop, shovel
Trier
Hard or packed wastes Drums, sacks, trucks Auger
192
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will depend on the characteristics of the solid matrix. It should
be continued until the material can pass through a 1-mm standard
sieve or can be extracted through a 1-mm diameter hole (USEPA 1982).
b. Contains volatiles - Subject it to an overnight evaporation at
approximately 35 Torr and room temperature in a vacuum manifold
(Guerin et al. 1980) or use distillation (Jones et al. 1977) or
evaporation (Guerin et al. 1980).
c. Contains liquids - Filter through a Whatman No. 42 filter paper.
\
Chemical Extraction— N
Choice of extraction solvents—Solvents that have been used to extract
organic material include n-pentane, benzene, tetrachlorobenzene, methanol,
dimethyl ether, methylene chloride, chloroform, carbon tetrachloride, toluene
and certain freons (Jobson et al. 1952, Giger et al. 1974, Kincannon 1972,
Patel 1974, Raymond et al. 1976, Walker et al. 1975, Sterns et al. 1968). The
choice of which solvent should be used can be based on safety, cost, solvent
properties and any specific requirements of the extraction procedure. For
example, trichlorotrifluoromethane is not very toxic and is slightly superior
to carbon tetrachloride in extracting oils when acid and salts are used, and
n-pentane does not extract phthalic compounds. However, it should be noted
that the USEPA standard for analytical chemical extractions is dichloromethane
(USEPA 1982). This solvent can be used to prepare samples of complex
mixtures, provided that it is removed before addition to the bioassay.
Extraction methods—
a. Soxhlet extraction method. This method allows for the extraction of
semivolatile and nonvolatile organic compounds from solid state
materials prior to fractionation. Basically, the method consists of
mixing the sample with anhydrous sodium sulfate in an extraction
thimble or between two plugs of glass wool and extracting with the
appropriate solvent in a Soxhlet extractor.
b. Sonication extraction method. The sonication method produces
disruption to ensure intimate contact of the sample matrix with the
extracting solvent. It suffers from the disadvantage of producing
artifacts and driving off some semivolatile compounds. Basically,
the method consists of mixing a ground solid in an extraction medium
and then dispersing it using sonication.
c. Blender extraction method. This is perhaps the simplest method of
extraction. Basically, it consists of placing a weighed amount of
ground sample in a blender together with the extracting solvent and
blending. It is a much simpler method than the other two.
Chemical Fractionation—
Chemical fractionation separates the crude extract into a number of
groups. The organic constituents of the extract are amenable to evaporation
193
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^ - 0
fractionation using chromatography? This senar! ^ f6" devel°Ped
individual compound classes (Miller 1982 * C°mpleX mixture i
et al. 1982, Crovley et al! 1%" Brocco
into
has been used in the conical fj
mixtures (Colgrove and Svec 1981)
Matsushita 1979, Novotnv et al
Guerin et al. 1980)1
extraction is the
2SSS o,
f Jf J f a variety of complex
p i " f?iels ^Buchanan 1982,
W et ' 1981< Epler et al- "78,
with an aqueous inorganic acid
;r"'"r="" -
complex mixture is
separaed Into
ml]!ture
*•
.rdk .„ P,ttiay
•
194
-------
and polymerization are likely to occur with time (Worstell and
Daniel 1981, Worstell et al. 1981). This may partially account for
the variability being detected by some investigators.
c Neutral organics. A variety of organic compounds are detected in
this group. These include aliphatic and aromatic hydrocarbons , as
well as oxygenated and chlorinated hydrocarbons. Some workers have
included additional separatory steps in their J™'^™"^
to further delineate this group (Epler et al. 1978, Guenn 1980).
These additional separations include using various columns and
eluting with different solvents.
d. Water-soluble. This group includes constituents which were not
solvent-extractable .
e. Residual solids. Included in this group are Inorganics and
materials such as coke.
It should be pointed out that some compounds may not survive the acid-
base fractionation because of hydrolysis. These include esters, halogenated
hydrocarbons, phthalates, nitriles and amides. Some compounds are altered to
other compounds. Included in this group are DDT, DDD and toxaphene (USEPA
1980c). in order to reduce the hydrolysis, Felton et al. (1981) used 0.01N
acid and base in their extraction procedure, instead of the more commonly used
X concentration. Still other workers have avoided the difficulty completely
£ usinrcolumn-based fractionation schemes (see below). However, Pelrov et
al (1981)! in side-by-side studies of liquid-liquid acid-base fractionation
*
and Sephadex LH-20, could detect no mutagenic M**™*™*^^
separatory schemes when examining synthetic fuels. Similarly, no difference
was detected for extraction of complex mixtures from cooked -eats ^e»
isopropyl alcohol or acetone was the solvent of choice (Felton et al. 1981).
Both of these extraction procedures were 90% efficient and it was suggested
that no artifacts were formed with either extraction method (Felton et al.
1981).
Other schemes of fractionation-These essentially include chromatographic
separation using procedures similar to those used to isolate J™^1*"1
compounds (see USEPA 1982). Except for those using Sephad ex LH- 20 gel and
different eluting solvents (Rao et al. 1981, Pelrov et al. 1981, ££• et^l.
1982), most of the other separation techniques were applied to specialized
situations, such as the analysis of polycyclic aromatic hydrocarbons in marine
organisms and sediment (Bjorseth 1979) or the rapid Tenax-GC extraction tech-
nique (Shiaras et al. 1980). High-pressure liquid chromatography has also
been used directly to determine impurities in carcinogens using the Ames test
(Donahue et al. 1978).
Although the standard Ames test or the preincubation modification would
be the methods of choice at this time, another method with limited application
uses thin-layer chromatography (TLC). This quick and direct method of detect-
ing mutagens by applying the Salmonella assay directly onto TLC plates has
lien wed to sLdy mutation associated with photocopier toner and typewriter
ribbons (Bjorseth et al. 1982). Others have used TLC plates by extracting the
spot (Pitts et al. 1978, Hayashida et al. 1976, Wilson et al. 1980, Issaq
195
-------
Chemical Isolation —
indivtduanfisoLS0^' ^^ Cartography Procedures may be used to
individually isolate compounds. The inclusion of these in the initial
Concluding Remarks
In nJlTT °. the fractlonation schemes for bioassays have not been verified
«1JZ< r T Cf8 thS meth°dS USed are simllar to ^ose already verified for
organic chemical analysis. The inorganic components have not beln examined in
baS? "rf UreS' alth°U?h ±C 1S knOWn that m*tals can -duce mutation in
bacteria (Kanematsu et al. 1980) and carcinogenesis (Saunderman 1978°?
iauldHn-H f ±nvolves blender extraction followed by acid-base
compounds^ *" ^ ?', Subse^uent identification of the individual
However it shouirr^,1 1 ^ SeParatiOn by Various ^hromatographic means.
However, it should be pointed out that as the number of fractions increases
nro±? T5er,°f b±0assays' T^S, more fractions raean more cost One
promising method that seems to alleviate this problem is the direct thin-laver
chromatography Ames test. In all instances, irrespective of the fractionatlon
carrXr^olvLr03,8837 USe^/he Varl°US fractionSare usually dissolved in T
?980 for- ^ frl°r /° addlt±on to the b±oassay (see Abbondandolo et al.
also be no. rr °K ** ^^ °f 13 Carrier s°lvents) . Finally, it should
also be pointed out that caution should be used in interpreting the result of
196
-------
REFERENCES
Abbondatidolo A, Bonatti S, Corsi G, Fiorio R, Leporlni C, Mazzaccaro A, Nierl
R, Borale R, Loprieno N. 1980. The use of organic solvents in mutagenicity
testing. Mutat. Res. 79 141-150.
Adams CE, Ford DL, Eckenfelder WW. 1981. Development of design and operation
criteria for wastewater treatment. Nashville, Tennesee: Enviro Press, Inc.
Adams J, Donnelly KG, Anderson DC* 1983. Hazardous waste streams. In:
Brown K, Evans GB, Frentrup BD., eds. Hazardous waste land treatment.
Chapter 5. Boston: Butterworth Press, pp. 127-163.
Bell JH, Ireland S, Spears AW. 1969. Identification of aromatic ketones in
cigarette smoke condensates. Anal. Chem. 41:2, 310-313.
Bjorseth A, Eidsa G, Gether J, Landmark L, Moller MM. 1982. Detection of
mutagens in complexed samples by Salmonella assay applied directly on
thin-layer chromatography plates. Science 215:87-89.
Bjorseth A. 1979. Determination of polycyclic aromatic hydrocarbons. In:
sediments and mussels from Saudafjord, W. Norway, by glass capillary gas
chromatography. The Science of the Total Environment. 13:71-86.
Boduskznski MM, Hurturbise RJ, Silver HF. 1982a. Separation of solvent-
refined coal into solvent derived fractions. Anal. Chem. 54:372-375.
Boduskznski MM, Hurturbise RJ, Silver HF. 1982b. Separation of
solvent-refined coal into solvent derived fractions. Anal. Chem. 54:375-381.
Brocco D, Citnmino A, Possanzini M. 1973. Determination of azaheterocyclic
compounds in atmospheric dust by a combination of thin-layer and gas chromato-
graphy. J. Chromatogr. 84:371-377.
Brown KW, Donnelly KG, Scott BR. 1982. The fate of mutagenic compounds when
hazardous wastes are land treated. In: Land disposal of hazardous waste.
Proceed. 8th Annual Res. Symp. EPA-600/9-82-002 383-397.
Brown KW, Donnelly KG, Scott BR. 1983. The fate of mutagenic compounds when
hazardous wastes are land treated. In: Land disposal of hazardous waste.
Proceed. 9th Annual Res. Symp. EPA-600/9-83-002 482-497.
Buchanan MV. 1982. Mass spectral characterization of nitrogen-containing
compounds with ammonia chemical ionization. Anal. Chem. 54:570-574.
197
-------
JffiV edhe rsent decre? on
wastewat
o.i 11 -d characterization
elm 52:?224*y ' ch— tography and capillary gas chromatography. Anal
s'forT^rLP' St6PhenS R°' ^ ^ 198°' ' SamPlers and "-"P
v Of f?3 f T ** ! Streams' Municipal Environmental Research
ois "Ch 3nd Devel°Pme^. Cincinnati, Ohio. EPA
Donahue EV, McCann J, Ames BN. 1978. Detection of mutagenic impurities in
Mi978 M' ?ard^ee1AA> Ra° TK, Guerin MR, Rubin IB, Ho CH, Clark
synthetif ' futltl^i *? bi°lr°*ical analysls °* test materials from the
S tvnhim,,^, ? hn0log1ies' Z- Mutagenicity of crude oils determined by the
1- typhimurium microsomal activation system. Mutat. Res. 57:265-276.
Felton JS, Healy S, Struermer D, Berry C, Timourian H, Hatch FT, Morris M
Bqeldanes LF. 1981. Mutagens from the cooking of food. I. Improved extrac-
-1011 °' ^ ^
>«*
GigerW, Rienhart M, Schafner C, Stumm W. 1974. Environ. Sci. Technol..
Grabow WOK, Burger JS, Hilner CA. 1981. Comparison of liquid-liquid extrac-
txon and resin adsorption for concentrating nmtagens in Ames Samonella/
microsome assays on water. Bull. Environ. Contam. Toxicol. 27:442-449.
Guerin MR, Ho CH, Rao TK, Clark BR, Epler JL. 1980. Separation and identifi-
a°m °f ***"»— «">•«**"• 'l-t.ro.. J. Environ.
Hayashida S, Wang CY, Bryan GT. 1976, Gann 67:617.
Issaq HJ, Andrews AW, Janini GM, Barr BW. 1979. Anal. Chem. 53:347.
Jobson A, Mclaughlin M, Cook FD, West lake DWS. 1952. Appl. Microbiol,
198
-------
Jones AR, Guerin MR, Clarke BR. 1977. Preparative-scale liquid chromato-
graphic fractionation of crude oils derived from coal and shale. Analyt.
Chem. 49:1766-1771.
Kanematsu N, Kara M, Kada T, 1980. Rec assay and mutagenicity studies on
metal compounds. Mutat. Res. 77:109-116.
Kincannon CB. 1972. Oily waste disposal by soil cultivation process. U. S.
Environmental Protection Agency, Prot. Tech. EPA -122-72-110.
Later DW, Lee ML, Bartle KD, Kong RC, Vasilaros DL. 1982. Chemical class
separation and characterization of organic compounds in synthetic fuels.
Anal. Chem. 53:1612-1620.
Lin DCK, Foltz RL, Lucas SV, Petersen BA, Slivon LE, Melton RG. 1979. Glass
capillary gas chromatographic-mass spectrometric analysis of organics in
drinking water concentrates and advanced waste treatment water
concentrates—II. In: Van Hall CE, ed. American Society for Testing And
Materials. ASTM STP 636, 68-84.
Lundi G, Gether J, Gjos N, Land MBS. 1977. Organic micropollutants in
precipitation in Norway. Atmos. Environ. 11:1007-1014.
Matsushita H. 1979. Micro-analysis of polynuclear aromatic hydrocarbons in
petroleum. Am. Chem. Soc. Div. Fuel Chem. 24:292-298.
Miller R. 1982. Hydrocarbon class fractionation with bond-phase liquid
chromatography. Anal. Chem., 54:1742-1746.
Novotny MJ, Strand W, Smith SL, Weisler D, Schwende FJ. 1981. Compositional
studies of coal tar by capillary gas chromatography/mass spectrometry. Fuel
60:213-220.
Patel MS. 1974. Anal. Chem. 46:794,
Pelrov RA, Schlarew DS, Downey SP. i'"cl» Comparison of mutagenicities of
fossil fuels. Mutat. Res. 90:233-245.
Pitts JN, Van Cauwen L, Berghe KA, Grosjean D, Schmid JP, Fritz DR, Belser WL,
Knudson GB, Hayes PM. 1978. Science 202:515.
Rao TK, Allen BE, Ramsey DE, Epler JL, Rubin IB, Guerin MR, Clark BR.
Analytical and biological analysis of test materials from the synthetic fuel
technologies. III. Use of Sephadex LH-d20 gel chromatography technique for
the bioassay of crude synthetic fuels. Mutat. Res. 85:29-39.
Raymond RL, Hudson JO, Jamison VW. 1976. Appl. Environ. Contain. Toxicol.
13:522.
Shiaris MP, Sherrill TW, Sayler GS. 1980. Tenax-GC extraction technique for
residual polychlorinated blphenyl and polyaromatic hydrocarbon analysis in
biodegradation assays. Appl. Environ. Microbiol. 5:165-171.
199
-------
f ' 5°?8,? ' *°rrison R- 1968« Oil spill: Decisions for debris
Materi*!* I°Ji i ; I En8ineers' Lon8 Beach, CA for Oil and Hazardous
Materials Spill Branch, Industrial Environmental Research Laboratory, Edison
N.J. U. S. Environmental Protection Agency, Contract No. 68-03-2200.
37?lt™0-46W' 1978' CarCinogenic eff"ts of metals. Federation Proceedings,
Toste AP, Sklarew DS, Pelrov RA. 1982. Partion chromatography-high-
performance liquid chromatography facilities the organic analysis and bio-
testing of synfuels. J. Chromatogr. 249:267-282.
USEPA. 1982. Test methods for evaluating solid waste: Physical /chemical
methods. 2nd ed. U. S. Environmental Protection Agency. SW-846. July, 1982.
Walker JDColwell KB, Hamming MP, Ford HT. 1975. Bull. Environ. Contam.
loxicol. 13: 245.
Wilson BW, Pelrov R, Cresto J.T. 1980. Mutat. Res. 79:1930.
Worstell JH, Daniel SR. 1981. Deposit formation in liquid fuels. 2 The
?n , Q? °!yselected compounds on the storage of Jet A turbine fuel. Fuel
oU : 4oi— 4o4 .
Worstell JH Daniel SR, Frauenhoff JJ. 1981. Deposit formation in liquid
tuels. 3. The effect of selected compounds on the storage of Jet A turbine
fuel. Fuel 60:485-487.
200
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WASTE SOLIDS WORKGROUP REPORT
Workgroup Tasks
Key Peer Review Comments—
The following points were raised during the initial review. These were
listed as an initial starting point in our discussion. It was noted that the
purpose of the meeting was to discuss sample preparation for use in bioassays,
and not what to do in the bioassay testing. Bioassays were only involved when
they affected the method of sample preparation.
1. What is the importance of the normality of the acid and base
solutions used in the liquid-liquid extraction?
2. What size should the particles be for the extraction procedure? It
has been suggested that 4 mm should be used instead of 1 mm.
3. What are the differences in the three extraction procedures
(Soxhlet, blender or sonication)? Is one more efficient and less
expensive than another?
4. What storage conditions should be used? Are there any problems or
limitations associated with this area of the protocol?
5. What procedure should be used for solvent reduction?
6. Should samples be treated with additional substances (e.g.,
inclusion of surfactants), and does this enhance the extraction
procedure?
7. What do we do about the inorganic chemicals? Where would they occur
in an acid-base fractionation scheme?
8. Do we need to include internal standards? Which chemicals should be
considered for inclusion? Is this an effective means to evaluate
procedures and interlaboratory reproducibility?
9. Are there any special problems associated with a particular group of
chemicals (e.g., weak mutagens)?
Issues Identified During the General Sessions and Workgroup Discussion—
Other points were raised during the general sessions. These are listed
as a continuation of the points under Key Peer Review Comments so that they
can be referred to later.
201
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' fr^M^ S ^ ^f rUCti°n °f muta8enic activity, should acid
fractionation be conducted prior to base fractionation?
11. Should acid, base and neutral fractions be subfractionated (e.g.,
weak, strong and precipitated acids)?
fractlonation Procedures have been adequately
mlnlmum and maximum number of fractions to be tested in
D1O3SS ciy •
14. What should be used as upper and lower limits of residue to be
tested in bioassay (mg/plate)?
protoco1 (what types of material
16. Should a crude extract using DMSO be tested as an initial sample?
17. How many replicates should be used?
18. What solvents are compatible with the bioassays?
19. What purity of solvents should be used?
20. Redefine waste solids to be compatible with other protocols.
Summary of Workgroup Progress —
A11 of the above issues were resolved as far as the available scientific
"—*'•» «-"«t ui-i.sj.iiaj. protocol was deemed
acceptable as a method for preparing samples of the solid matrix for use in
follows: n8' abolishments of the workgroup can be summarized as
a. All of the modifications made to this protocol were aimed at
reducing degradation of the complexed mixture. The major
modifications were:
• Dissolve the complexed sample in the solvent before addition of
the acid or base.
• Use an acid fractionation before a base fractionation.
• Conduct all extraction, where possible, at 4 C.
b. An overall scheme of sample preparation and testing was devised.
The final definition (Issue 20) was adjusted to be compatible with
the definitions for the other types of matrices. This definition
TJ£) O •
c.
was
202
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A solid waste or solid complexed mixture, obtained from different
sources, is defined as that waste or complexed mixture containing
less than 50% water and having relative firmness, coherence of
particles or persistence of form as matter that is not liquid or
gaseous at 25 C. In the present document, it will be considered to
include tarry materials that are sticky or viscous (such as coal tar
sludge and adhesives wastes), airborne particulates in a gaseous
media and solids partitioned from wastewaters or other fluids.
Summary of Key Workgroup Discussions
Major Consensus Opinions—
Conditional statements—
a. It is recognized that liquid-liquid acid-base extraction has the
potential of altering components of complexed mixtures and environ-
mental samples. When the objective is an estimation of the overall
mutagenicity of the sample, the use of alternative extraction and
separation procedures (e.g., HPLC; see Appendix 3) does not appear
warranted based on currently available relevant scientific informa-
tion.
b. Because of the varied nature and sources of the waste solid cate-
gory, refinement of the existing protocol may be deemed necessary at
some future time.
Ngn-issues (Issues 7 and 17)—In general session, it was generally agreed
that analysis of vapor-phase organics and inorganics was not to be considered
in the present protocol. Similarly, the number of replicates required will be
dictated by the statistical design of the experiment.
Overall extraction and testing scheme (Issues 13, 14 and 18)—To simplify
the decision processes involved in the estimation of the mutagenicity of com-
plexed mixtures and environmental samples, the following scheme was devised.
Ideally, the volume of addition to mutagenicity test plates should be
held at 50 yL per plate. The dose range considered was 10 ng to 10 mg/plate
for nontoxic materials. If a positive nontoxic response is observed in this
dose range, no further testing is required. Methods considered for evaluation
of toxicity include reduction of the bacterial lawn, zone of growth inhibition
in spot tests and calculations of percent survival using an isogenic strain
currently being developed for toxicity estimation (Mortelmans K., Mutation
Research 1983).
If a toxic response is observed or a negative mutagenicity result is
obtained, further fractionation into acid, base and neutral fractions is
indicated. In the case of the negative response, such a fractionation is
optional and dependent on the experimental design. To minimize the cost of
sample preparation for bioassay evaluation, no further fractionation was
considered to be warranted unless additional chemical characterization or
mutagenicity of individual groups of compounds was required.
203
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than 2 ™ <""> considered
f- ^w-ssi
addition of dry Ice or by freeing samplea and ?hen
c.
U • r/UES i_ Tiam r> i aa -i -n nmi*. -I ^_ *._.*„
be included
-uxxowing me procedures for isolation of airborne
discussed in the Air Sample Preparation Protocol
* ^ ' *• *• u**c cALzracts ana i r* i T*TT»/*^ooa/i ^-«*-*.«.« — *. -m _ _ .
considerations, stora.e
As a general rule, preservatives should be excluded
Extraction procedures (Issues 3, 5. 6. 16 and 1 9 ) —
a> 2frS°5let 6X5rf t±0n technl^e ^s selected as the principle
Adams 1983 and in the section on Soils and Sediments).
204
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Inclusion of additional substances (e.g., surfactants) should be
avoided, except where scientific data support their inclusion in
order to obtain a greater efficiency of extraction.
b. The solvent selected for extraction was methylene chloride. Dimethyl
sulfoxide (DMSO) was not selected for the initial solvation because
of possible interferences with chemical analysis and the possibility
of a less efficient extraction procedure. The purity of the extract-
ing solvents should be spectroscopic grade or higher.
c. Apparatus for solvent reduction - Although the rotary evaporator may
be preferable for particular complexed mixtures, the Kuderna-Danish
apparatus described in the protocol will be maintained in order to
utilize equipment currently in use in commercial laboratories and to
facilitate processing multiple samples.
An aliquot of crude extract will be taken to dryness and redissolved in
DMSO at a concentration of 200 mg/mL (or the highest solubility). Although
DMSO will be considered as the solvent of choice for bioassay, other solvents,
including water, 10% ethanol or acetone, may be used depending on the sample
type.
Fractionation procedures (Issues 1. 4, 10 and 11)—
a. Normality of solutions - 1 Normal solutions of acids and bases will
be utilized to avoid compound destruction. Bases and acids are
added to the sample dissolved in methylene dichloride (or the
extracting solvent). The acid separation is also to be undertaken
before the base separation to limit the degradation of compounds.
Likewise, it is suggested that these separations be undertaken at
4 C.
b. No separation into weak, strong and precipitated (acid, base or
neutral) fractions was considered necessary.
c. Only the back-extractable acids, bases and neutral compounds will be
evaluated.
d. Hydrochloric acid is preferred to sulfuric acid, as the former
causes less decomposition than the latter.
Standardization of procedure (Issue 8)—The only standard on which
consensus agreement has been reached is a field blank to ensure that artifacts
are not introduced by the extraction procedure.
The selection of additional standards to monitor the extraction procedure
and to assess interlaboratory reproducibility was considered; however, the
chemical species were not designated. It was recommended that choice of
chemicals should be representative with respect to chemical class and extrac-
tion properties. The value of the addition of a spike was questioned because
of the potential for chemical reactivity with other components of a complex
mixture and for possible antagonistic or synergistic responses in a bioassay.
205
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Other Issues —
In addition to the unresolved issues raised in the section on Mai or
Consensus Opinions due to the lack or the limited nature of a scientific data
base, various issues of a general nature should be considered:
a. The application of the designated procedure to this category has not
been validated when the objective is the preparation of sample for
b. Although vaper-phase and inorganics have been excluded for general
consideration, the workgroup has questioned whether the elimination
of these chemicals from consideration is appropriate for the waste
solid category. Heavy metals are known to exert a mutagenic
response in bioassay systems other than the Ames test, and some
connections have been established between heavy metals and
carcinogenesis. As for the vapor-phase organics, these produce
responses in the Tradescantia system and the dessicator assay.
c. Although not directly discussed in the present meeting, it was
considered important that other short-term tests, in addition to the
Ames system, be used. Several reasons for this are that regulatory
decisions are seldom made on the basis of a response in a single
system and that additional short-term tests which have the
capability of detecting genetic damage other than "mutation" (e.g.,
chromosome changes, spindle damage and DNA repair capacity) should
also be employed. The genotoxic agents may not induce mutation, but
may be very effective in inducing aneuploidy. Such a genetic
alteration has been linked to cancer and birth defects.
d. Use of chromatographic methodology as an alternative sample
fractionation was considered to have numerous disadvantages,
including:
• Massive production of solvents
• Expertise required
• Increased cost associated with preparation of sample for
mutagenicity testing
• Maximum number of samples per equipment per working day appears
to be about eight
However, some members of the general session believed that this method is
superior to the liquid-liquid acid-base fractionation scheme.
e. Best approach - With respect to percent chemical recovery, the
protocol may not be the most cost effective or practical from the
viewpoint of equipment, expertise and the number of samples which
can be processed under given time restraints. These questions can
be effectively resolved by comparative experimentation with the
different methodologies in a cost effectiveness and efficiency
study. J
206
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Evaluation of Proposed Protocol
The protocol was evaluated in workgroup and In the general session. The
opinions of both groups coincided and are summarized below.
Adequacy—
As a first line basis for preparing samples for testing in bioassays, the
protocol was considered adequate. However, it was noted that for several
crucial areas, such as sample storage or the best method of extraction, a
broad data base was unavailable for formulating a scientific decision.
Validity--
One of the main limitations of the waste solid protocol is that the
method has only been used for a limited number of samples in a small number of
labs. It is assumed that because the methods are, in essence, those used for
chemical analysis of organic complex mixtures and environmental samples, they
have been well-documented for this type of analysis.
Limitations—
Besides the obvious limitations of the methods to those organics that are
back-extractable to the solvent and remain unaltered at the time of assay, one
of the limitations of this protocol is the lack of an adequate data base for
formulating a specific protocol.
Comparison to Other Protocols—
Similar limitations due to the absence of a data base were also apparent
in all but one protocol. The methodologies suggested are comparable to those
used for the other media.
Other Data or Information Requirements
Information Gaps—
As indicated above, numerous gaps exist in the scientific data base.
These have been divided into major and minor gaps. The major gaps are those
questions that are considered necessary to be answered before the protocol can
be utilized in a decision making process. Those that are considered minor
would improve the efficiency of the protocol and increase the confidence in
the data obtained with this process by determining the limitations of each
method.
Major gaps—
1. Storage conditions - This is a prerequisite of any of the prepara-
tive protocols. Although some data are available on the storage of
complex mixtures and environmental samples, no definitive study is
available concerning the effect of storage temperature and time on
the stability of the samples for analysis with a bioassay system.
2. Validation of the overall protocol - The process for deciding which
way to proceed with the extraction and separation procedure was
207
-------
f°r thl8 "'""co1- Th-- « is currently
sr-
Minor Raps —
1tations of
extraction methods - Only
to Uquld-U,uld acid-
Research Program Needs —
=53 HriLlv rS
extractable, base-extractable, detected in the neutral fraction direct-
liquid liquid acid-base fractionation could be made.
invest iLtion°n J,lsadvan^?f . to utilizing synthetic mixtures in this type of
evllultld 6 P°SSlblllty tha< not all the various permutations Sll be
208
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PROTOCOL FOR THE PREPARATION OF WASTE SOLIDS FOR MUTAGENICITY TESTING
1.0 Scope and Application
Complexmixtures may be composed of a diverse mixture of organic and
inorganic compounds present in a variety of matrices (i.e., solids, liquids
and gases). They can be obtained from such widely different sources as the
environment or manufacturing and treatment processes. The following document
presents the best protocols currently available to genetic toxicologists for
sample preparation for this matrix.
The methods are limited to those organics that are extractable by the
extracting solvent (normally dichloromethane).
2.0 Summary of Method
To simplify the decision process involved in estimating the muta-
genicity of complexed mixtures and environmental samples, the following scheme
was devised.
Ideally, the volume of addition to mutagenicity test plates should be
held at 50 yL per plate. The dose range to be assayed should be 10 ng to
10 mg/plate. If a positive nontoxic response is observed in this dose range
with the crude solvent-extract material, no further testing is required.
If a toxic response is observed or a negative mutagenicity result is
obtained, further fractionation into acid, base and neutral fractions is
indicated. In the case of the negative response, such a fractionation is
optional and dependent on the experimental design. To minimize the cost of
sample preparation for bioassay evaluation, no further fractionation other
than that indicated above is considered to be warranted unless additional
chemical characterization or mutagenicity of individual groups of compounds is
required.
3.0 Definitions
A solid waste or solid complex mixture, obtained from different
sources, is defined as that waste or complex mixture containing less than 50%
water and having relative firmness, coherence of particles or persistence of
form as matter that is not liquid or gaseous at 25 C. In the present
document, it will be considered to include tarry materials that are sticky or
viscous (such as coal tar sludge and adhesives wastes), airborne particulates
in gaseous media and solids partitioned from wastewaters or other fluids.
209
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4.0 Safety
P°Jential hazards °f the solid complex mixture are known.
Tny lnstances thls is not the case. Workers should assume
f?±*M T T < tOX±C ^ Presents other da«gers, such as explosive or
flammable tendencies. Each of these hazards should be handled in the
appropriate manner, (see Steere 1971, Sax 1965, Bretherick 1975, USEPA 1977
5.0
Sample Collection. Preservation and Handling
Equipment and procedures have already been established and described
n^iS°J matrlX SampleS (see de Vera et a1' 1980>- ^ese are
in Table 6. The precautions taken for samples to be analyzed for
organic analysis are adequate for samples to be prepared for use in the
bioassays (see de Vera et al. 1980).
of ltffhfUn-rS th! "mPlex mlxt«res are known to be unreactive in the presence
of light, it is advisable to perform manipulation under reduced light or
yellow lighting. 6
not pii-Ji'vi8 !"°8nlzed *ha5 ^intenance of the sample in a state which does
If the nroto^ 1 S"7 * * T ^ ±B * necessary Prerequisite for success
of the protocol. Three areas of storage are to be considered: (a) original
samples of the bulk matrix, (b) crude extracts and (c) processed extract. For
practical considerations, storage conditions will be influenced by sample size
and number.
When feasible, maintenance of temperatures at 4 C or less should be
considered for all samples, and ideally, samples (b) and (c) should be stored
at -20 C or less.
As a general rule, preservatives should be excluded.
Samples should be stored in amber glass containers with Teflon caps.
in the case of glass-corrosive substances (e.g., hydrofluoric acid), an
alternative container made of Teflon is recommended.
6.0 Calibration
Blank samples demonstrating that the methods used did not contaminate
the sample should be run with each batch of samples prepared.
The number of replicates required will be dictated by the statistical
design of the experiment.
7.0 Quality Control
Laboratories preparing samples of waste "solid" should adhere to the
Guides for Quality Assurance in Environmental Health Research and the proposed
good laboratory practices" (FDA 1976, 1977, 1978, USEPA 1980). Not only
should these practices take place in the laboratory, but they also should be
210
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practiced at the point of sampling and transportation. This means that good
scientific practices and good administrative record keeping and labeling (see
Wastewater Protocol for example of record sheets) should be employed. It is
vital that all materials used in the preparation of the sample for bioassay
analysis be of spectral grade or equivalent (see de Vera et al. 1980 for
further details). The glassware and equipment should also conform to the
standards established for cleaning for water and wastewater (de Vera et al.
1980).
8.0 Procedure
8.1 Extraction— \
The solid matrix may contain volatiles or be contained in a liquid
matrix. Before any material can be used for extraction, the contaminating
matrices must be removed. Sufficient,,material to yield approximately 40 g of
solid should be used in each case. This is the raw material to be extracted.
Because of the heterogeneous nature of the material included in this
category, the following methods of physical separation may be considered:
a. Reduce particle size to less than 2 mm by grinding.
b. When dealing with oily, gummy or adhesive wastes, additional
treatment with anhydrous sodium sulfate or fumed silica gel
(Caba-o-sil Corp., Boston, Mass.) may be warranted. Alterna-
tively, size reduction for these materials may be accomplished by
the addition of dry ice or by freezing the samples and then
grinding with dry ice.
c. Liquids can be separated from solids by gravity phase separation
for 24 h at 4 C (see Wastewater Protocol).
d. Dust particles in ambient air are also to be included in this
protocol. Follow the procedures for isolation of airborne
particulates discussed in the Air Sample Preparation Protocol.
8.2 Soxhlet Extraction Method—
8.2.1 Scope and aplication—This method provides a procedure for extracting
organic compounds from a solid matrix prior to fractionation for use in a
bioassay.
8.2.2 Summary of the method—The solid sample of <2 mm particulate size is
mixed with sufficient sodium sulfate until dry, placed in an extraction
thimble or between two plugs of glass wool and extracted using an appropriate
solvent in a Soxhlet extractor. The extract is then concentrated and used in
the fractionation procedure.
8.2.3 Apparatus and equipment—
a. Soxhlet extractor - 40-mm ID, with 500-mL round-bottom flask.
211
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c. Chromatography column - Pyrex, 20-mm ID, approximately 400 irnn
^£tr^^^
8.2.4 Reagents and consumable materials—
s Acs (purified
8.2.5 Procedure —
s
glass woo. r 3n ten C°Ver W±th anoth- Plug of
b. Plac* 300 mL of the solvent into a 500-mL round-bottom flask
containing a boiling stone (solvent-extracted); attach the flask
to the extractor and extract the solids for 16 h.
' fllter and
Wlth a 10-mL *raduated concentrator
cenraor
to W5 28of e"raCt° flask and sodi^ -«««te column with 100
usine the K D extractin8 sol^nt. It is then concentrated
using tne K-D apparatus.
d-
HS'T^^ -—
of Slfi %COnCeJtrut0r tUbe and the ent±re lower '««nded surface
of the flask are bathed in hot water or vapor.
t^!!r ?6 Vert±Cal P°sltion of the equipment and the water
temperature to complete the concentration in 15 to 20 min
b^rl11^617 f CK At the pr°Per rate of distillation,
aCtl
not rlod » u e <«
not flood. When the volume of the liquid reaches 1 to 2
apparatus
212
-------
e. Transfer with solvent washes to a tared sample tube. Dry under
nitrogen and weigh residue.
8.3 Blender Extraction Method—
8.3.1 Scope and application—This method provides a procedure for extracting
organic compounds from a solid matrix prior to fractionation for use in a
bioassay. It is quicker than the standard Soxhlet method. Both methods
produce a sample for further fractionation.
8.3.2 Summary of the method—The solid sample of 1 mm particulate size is
mixed with sodium sulfate, placed in a blender and extracted using an
appropriate solvent. The extract is then concentrated and used in the
fractionation procedure.
8.3.3 Apparatus and equipment—
a. Blender with a screw cap (explosion-proof type suggested).
b. Kuderna-Danish apparatus with a three-ball Snyder column or vacuum
rotary evaporator with a chilled solvent receiving flask.
c. Chromatography column - Pyrex, 20-mm ID, approximately 400 mm
long, with a coarse fritted plate at the bottom and an appropriate
packing medium. This is used as a drying column.
8.3.4 Reagents and consumable materials—
a. An appropriate solvent or solvent mixture of spectroscopic grade.
In most instance, dichloromethane can be used.
b. Anhydrous sodium sulfate, ACS (purified by heating at 400 C for
4 h in a shallow tray).
8.3.5 Procedure—
a. Mix 10 g of solid with 10 g of anhydrous sodium sulfate and place
in the blender.
b. Add 300 mL of the solvent into the blender. This should half fill
the jar. Screw jar cover onto the blender jar tightly.
c. Holding the lid of the blender, blend for 30 sec. Wait 5 min and
blend for 15 sec. Repeat the last step.
d. When complete, filter using a Whatmans No. 41 filter paper and dry
it by passing it through a 4-in column of sodium sulfate which has
been washed with the extracting solvent. Collect the dried
extract in a 500-mL K-D flask fitted with a 10-mL graduated
concentrator tube. Wash the extractor flask and sodium sulfate
column with 100 to 125 mL of the extracting solvent. It is then
concentrated using the K-D apparatus.
213
-------
f • to
8.4 Llqutd-Ltquld Extractlon-Fractionatlon—
"dure
-
and
separated fract^rfurther'see'sectionTs)?068' " *** be necessary~to
8.4.3 Apparatus and equipment—
a. Separatory funnel with Teflon stopcock (2,000 mL).
D • JS.UQ£TTlfl«—I)an-i oVi •****« **•«.«*. j ,.«
with a
c.
d. Boiling chips, solvent-extracted, approximately 10/40 mesh.
e. Water or steam bath.
8.4.4 Reagents and consumable materials—
a. Sodium hydroxide - (ACS) IN in distilled water (chilled to 4 C).
D • AiyujLuuii-Luinr' am n — fflr*c^ i *r t , . .
mixing concentrated HC1
214
-------
c. An appropriate solvent or solvent mixture of spectroscopic grade.
In most instances, dichloromethane can be used.
d. Anhydrous sodium sulfate, ACS (purified by heating at 400 C for 4
hours in a shallow tray).
e. Distilled water.
8.4.5 Procedure—
a.
Dissolve 10 g of the extract in 30 mL of solvent. In most
instances, the solvent will be dichloromethane.
b. Place in a separatory funnel
c.
a
Adjust the pH to 1-2 with HC1 (approximately 100 mL). Partition
the sample into the two phases by shaking the funnel for one
minute with periodic venting to release pressure (point funnel
away from the analyst).
d. Allow the fractions to separate in an upright stand (10-min
minimum). If the emulsion interface between layers is more than
one-third the size of the solvent layer, the analyst must employ
mechanical device to complete the phase separation. The optimum
mechanical technique depends upon the sample, but may include
stirring, filtration of the emulsion through glass wool or
centrifugation.
e The organic layer will be the lower phase. This phase will
contain the acidic and neutral fraction. Collect the upper phase
in a flask and label. This phase contains the base.
f. Reextract the solvent layer twice more with IN HC1 at pH 1-2
(approximately 50 mL each). Combine the aqueous base extracts.
g. Adjust the pH of the aqueous layer obtained in Steps e and f to pH
12-13 by addition of IN NaOH and extract the base fraction with
200 mL of dichloromethane.
h. Allow the fractions to separate in an upright stand (10-min
minimum). If the emulsion interface between layers is more than
one-third the size of the solvent layer, the analyst must employ a
mechanical device to complete the phase separation.
i. The organic layer will be the lower phase. This phase will
contain the base fraction. Collect the lower phase in a flask and
label. This phase contains the base fraction in dichloromethane.
Add 100 mL of dichloromethane and repeat the separation to collect
a second base fraction in dichloromethane, which is added to the
first.
215
-------
n.
o.
l^SV^S ?f th* S°1Vent Uyer obtained in Steps e and f to pH
the" sample i r .^ ™ *?* <«PP«**»tely 200 mL?. Partition ?
cne sample into the two phases by shaking the funnel for one
to release pressure
*• s:«sr±ti.T^jj^ are concen-
Corresponding aqueous fractions and any
represent the
wuu«uuir«ion is accomplished by adding one to two solvent-treated
boiling chips to the flask and attaching a three-balled Snyder
column Prewet the Snyder column by adding about 1 mL of solvent
so that the concentrator"tubePanatthS ^ " ***** °* ^ """ ba^
of the flask arrLtherirhotawfter\rvapor!°ldjustndhf ver-'"
rn^i J08"lon of the equipment and the water temperature to
complete the concentration in 15 to 20 min (approximately 45 C).
At the proper rate of distillation, the balls of the column will
216
-------
actively chatter, but the chamber will not flood. When the volume
of the liquid reaches 1 to 2 mL, remove the K-D apparatus and
allow to drain for at least 10 min while cooling.
t. Transfer with solvent washes to a tared sample tube. Dry under
nitrogen and weigh residue for the base, acid and neutral
fractions. These fractions can be tested in the bioassay.
8.5 Chemical Isolation—
This step is only necessary if the fractionation scheme giving base,
acid, and neutral fractions is insufficient to reduce the toxicity to a level
where the mutagenicity can be examined by the bioassay system or if the
identity of the individual compounds is required. If this is the case,
standard procedures are already established for chemical analysis that are
compatible with the biological systems (see EPA 1982a).
9.0 Calculations
The type of calculations used will depend upon the source of the
sample and the experimental design. For instance, the original bulk sample
could be separated into different matrices, each of which follows a separate
extraction-fractionation scheme. The samples obtained from each of these
schemes could then be composited (e.g., crude extract from a liquid matrix
combined with the crude extract from a solid matrix) or treated separately for
analysis by the bioassay system.
Irrespective of the experimental design, for each stage of the
extraction-fractionation scheme, the dry weight of each component should be
recorded (e.g., weight of original bulk sample, crude extract, acidic fraction
and neutral fraction). The genetic event for each bioassay can then be
calculated using standard methods accepted for each assay (see Williams and
Preston 1983 for details for the Ames test). It is suggested that the
response of each bioassay be expressed as genetic event/mg weight (e.g.,
revertants/plate/mg for the Ames system) of the sample fraction under
consideration.
10.0 Precision and Accuracy
Many of the fractionation schemes for bioassays have not been verified.
In most instances, the methods used are similar to those already verified for
organic chemical analysis. The most simple scheme involves blender extraction
followed by base-acid liquid-liquid fractionation. Subsequent identification
of the individual compounds can be accomplished by separation by various
chromatographic means. However, it should be pointed out that as the number
of fractions increases, so does the number of bioassays. Thus, more fractions
mean more cost.
Finally, it should also be pointed out that caution should be used in
interpreting the result of any bioassay using fractionated solid complex
mixtures, as various synergistic and antagonistic mutagenic effects occur to
different extents in the different fractions.
217
-------
REFERENCES
°f reaCU'e <*- «1 "-«*. Cleveland, Ohio
. pr°perties °f Austria! materials, 2nd ed., New
C^Prels. 19?1' Handb°°k °f laboratory safety. 2nd ed., Cleveland, Ohio:
USEPA 1977. Hazardous waste management facilities in the United States
t
USEPA 1980. U. S. Environmental Protection Agency. Part IV
8°°d ^"^ P-ctlcessda d for
and ecologlcal effects
S" ^1°'? '?' eValua5ln^ solid w-te: Physical /chemical
ed., U. S. Environmental Protection Agency. SW-856.
Saml]«/M.PreSt°n1JE- 1983' Interlm Procedures for Conducting the
Salmonella/Mxcrosomal Mutagenicity Assay (Ames Test). EPA-600/4-82-068
Environmental Monitoring Systems Laboratory, Las Vegas NV
218
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APPENDIX 1
TR-506-105B
LIST OF PARTICIPANTS
MUTAGENICITY SAMPLE PREPARATION PROTOCOLS
PANEL MEETING
AIR
Dr. Peter Flessel
Air and Industrial Hygiene Laboratory
California Department of Health Services
2151 Berkeley Way
Berkeley, CA 94704
(415) 540-2475
(a)
Dr. Marvin S. Legatorv
Division of Environmental Toxicology
Department of Preventative Medicine
and Community Health
University of Texas Medical Branch
Route F-17
Galveston, TX 77550
(409) 761-1803
Dr. Ray Merrill
Industrial Environmental
Research Laboratory (MD-60)
Research Triangle Park, NC 27711
(919) 541-2558
Dr. V. M. S. Ramanujam
Division of Environmental Toxicology
Department of Preventive Medicine
and Community Health
University of Texas Medical Branch
Route F-17
Galveston, TX 77550
(409) 761-3614
DRINKING WATER
Mr. Rodger B. Baird
Sanitation Districts of
Los Angeles County
San Jose Creek Water
Quality Laboratory
1965 Workman Mill Road
Whittier, CA 90601
(213) 685-9572 Ext. 215
Dr. Fred Kopfler
Andrew W. Breidenbach
Environmental Monitoring
Systems Laboratory/ORD
26 West St. Glair St.
Cincinnati, OH 45268
(513) 684-7451
Dr. M. Wilson Tabor(
University of Cincinnati
Medical Center
Institute of Environmental Health
Cincinnati, OH 45267-0056
(513) 872-4830
Dr. David T. Williams
Health and Welfare Canada
Tunney's Pasture
Ottawa, Ontario, Canada K1A-OL2
(613) 992-6812
continued-
(a) Indicates provided written review comments but not an attendee.
(b) Indicates Workgroup Leader.
219
-------
NON-AQUEOUS LIQUID WASTES
Dr. Stanton L. Gerson
Assistant Professor of Medicine
Division of Hematology and Oncology
University Hospitals of Cleveland
Case Western Reserve University
School of Medicine
2074 Abington Road
Cleveland, OH 44106
(216) 884-8532
Dr. Michael Guerin
Analytical Chemistry Division
Oak Ridge National Laboratory
P.O. Box X
Oak Ridge, TN 37831
(615) 574-4862
Dr. Donald Gurka
Environmental Monitoring
Systems Laboratory/ORD
P.O. Box 15027
Las Vegas, NV 89114
(702) 798-2100
Dr. Paul C. Howard
Center for Environmental Heath Science
Case Western Reserve University
School of Medicine
Cleveland, OH 44106
(216) 368-3439
Dr. Ronald J. Spanggord
SRI International
Pharmaceutical Analysis Department
333 Ravenwood Avenue
Menlo Park, CA 94025
(415) 326-6200
Dr. Yi Wang(b)
Air and Industrial Hygiene Laboratory
California Department of Health Services
2151 Berkeley Way
Berkeley, CA 94704
(415) 540-2475
SOILS AND SEDIMENTS
Dr. Kirk Brown
Texas A & M University
Suite 202, Bldg. D
707 Texas Avenue
College Station, TX 77840
(409) 845-5251
Dr. Ken Loveday
Bioassay Systems Corporation
225 Wildwpod Avenue
Woburn, MA 01801
(617) 933-9229
Dr. Paul Marsden
Lockheed EMSCO Inc.
P.O. Box 15027
Las Vegas, NV 89114
(702) 798-2100
Mr. Vincent I. Mastricola, Jr.
Millipore Corporation
80 Ashby Road
Bedford, MA 01730
(617) 275-9200
WASTE SOLIDS
Mr. K. C. Donnelly
Soil & Crop Science Department
Texas A & M University
College Station, TX 77843
(409) 845-5251
Dr. Elena C. McCoy
Center for Environmental Health
Science
Case Western Reserve University
School of Medicine
Cleveland, OH 44106
(216) 368-5962
Dr. Barry R. Scott
Phoenix-Lone Oak Laboratory
129 Bluff Street
P.O. Box 744
Smithville, TX 78957
(512) 237-4110
continued-
220
-------
WASTE SOLIDS - continued
OTHER PARTICIPANTS
Mr. Bart Simmons
Hazardous Materials Laboratory
California Department of Health Services
2151 Berkeley Way
Berkeley, CA 94704
(415) 540300
WASTEWATER
(b)
Dr. David J. Brusick
Litton Bionetics, Inc.
5516 Nicholson Lane
Kensington, MD 20895
(301) 881-5600
Dr. Dick Garnas
National Enforcement
Investigations Center (NEIC)
Building 53, Box 25227
Denver, CO 80225
(303) 234-4650
Dr. Ray Merrill
Industrial Environmental
Research Laboratory (MD-60)
Research Triangle Park, NC 27711
(919) 541-2558
Dr. T. Kameswar Rao
Environmental Health Research
and Testing, Inc.
P.O. Box 12199
Research Triangle Park, NC 27709
(919) 541-2342
Dr. Gary D. Stoner
Department of Pathology
Medical College of Ohio
3000 Arlington Avenue
Health Education Bldg. Rm. 202
Toledo, OH 43699
(419) 381-4408
U.S. Environmental Protection
Agency
Dr. Llewellyn Williams
Environmental Monitoring Systems
Laboratory
P.O. Box 15027
Las Vegas, NV 89114
(702) 798-2138
\
U.S. Department of Health and
Human Services
Dr. William M. Wagner
Centers for Disease Control
1600 Clifton Road
Atlanta, GA 30333
ICAIR. Life Systems, Inc.
Ms. Jo Ann M. Duchene
Mr. Jon P. Hellerstein
Mr. Jeffrey S. Beaton
Ms. Cynthia D. Patrick
OBSERVERS
Dr. Gordon Newell
Electric Power Research Institute
Environmental Assessment Division
P.O. Box 10412
Palo Alto, CA 94303
(415) 855-2573
Dr. Blakeman Smith
Electric Power Research Institute
Environmental Assessment Division
P.O. Box 10412
Palo Alto, CA 94303
(415) 855-2573
221
-------
APPENDIX 2
TR-506-106
AGENDA
MUTAGENICITY SAMPLE PREPARATION PROTOCOLS
PANEL MEETING
July 23-25, 1984
Hyatt Rickeys
Palo Alto, California
EPA Task Manager: Dr. Llewellyn Williams
Time
Monday,
July 23, 1984
8:00 a.m.
8:15 a.m.
9:00 a.m.
9:20 a.m.
9:50 a.m.
Agenda
Welcome
Administrative Announcements
2,
3.
4.
5.
Panel Meeting Overview
1. Background
Objectives
Assumptions
Approach and Schedule
Introduction of Workgroup Leaders
Presentations of Sample Preparation Protocols
1. Summary of Air Protocols
• Review paper
• Group discussion/recommendations
• Summary of group discussion and
workgroup tasks
Individual
J. Heaton
J. Heaton
L. Williams
V. Ramanuj am
L. Williams
V. Ramanujam
continued-
T^aneTme^HnFobservers are welcome to participate in all discussion
periods and workgroup meetings. Degree of observer participation in
workgroup meetings to be determined by Workgroup LeaSer ?
222
-------
Appendix 2 - continued
Time
Agenda Item
Individual
Monday -
continued
10:00 a.m.
10:20 a.m.
10:50 a.m.
11:00 a.m.
11:20 a.m.
11:50 a.m.
12:00 Noon
1:30 p.m.
1:50 p.m.
2:20 p.m.
2:30 p.m.
2:50 p.m.
3:20 p.m.
3:30 p.m.
3:50 p.m.
4:20 p.m.
4:30 p.m.
5:00 p.m.
Summary of Drinking Water Protocols
• Review paper
• Group discussion/recommendations
• Summary of group discussion and
workgroup tasks
Summary of Wastewater Protocols
• Review paper
• Group discussion/recommendations
• Summary of group discussion and
workgroup tasks
Lunch
4. Summary of Nonaqueous Liquid Wastes
Protocols
• Review paper
• Group discussion/recommendations
• Summary of group dicussion and
workgroup tasks
5. Summary of Soils and Sediments
Protocols
• Review paper
• Group discussion/recommendations
• Summary of group discussion and
workgroup tasks
6. Summary of Waste Solids Protocols
• Review paper
• Group discussion/recommendations
• Summary of group discussion and
workgroup tasks
Initial Workgroup Meetings
Adjourn for Day
W. Tabor
L. Williams
W. Tabor
D. Brusick
L. Williams
D. Brusick
R. Spanggord
L. Williams
R. Spanggord
K. Brown
L. Williams
K. Brown
B. Scott
L. Williams
B. Scott
Workgroup
Members
continued-
223
-------
Appendix 2 - continued
Time
Tuesday,
July 24, 1984
8:00 a.m.
8:15 a.m.
10:30 a.m.
10:45 a.m.
11:00 a.m.
11:15 a.m.
11:30 a.m.
11:45 a.m.
12:00 Noon
1:30 p.m.
3:30 p.m.
3:45 p.m.
Introductory Remarks
Workgroup Meetings
1. List workgroup tasks
2. Begin revising protocols per
group discussion
3. List unresolved issues
Workgroup Status Reports
• Status of workgroup tasks
• Unresolved issues
• Group discussion
• Revised tasks for workgroup
1. Status of Air Workgroup
2. S.tatus of Drinking Water Workgroup
3. Status of Wastewater Workgroup
4. Status of Nonaqueous Liquid Wastes
Workgroup
5. Status of Soils and Sediments
Workgroup
6. Status of Waste Solids Workgroup
Lunch
Workgroup Meetings
1. List revised workgroup tasks
2. Continue revising protocols per
group discussion
3. Resolve issues
Workgroup Status Reports
• Status of workgroup tasks
• Unresolved issues
• Group discussion
• Revised tasks for workgroup
1. Status of Air Workgroup
2. Status of Drinking Water Workcrrmm
Individual
L. Williams
Workgroup
Members
•
V. Ramanujam
W. Tabor
D. Brusick
Y. Wang
K. Brown
B. Scott
Workgroup
Members
V . Ramanuj am
TJ TnV««.
224
continued-
-------
Appendix 2 - continued
Time
Tuesday -
continued
4:00 p.m.
4:15 p.m.
4:30 p.m.
4:45 p.m.
5:00 p.m.
Wednesday,
July 25. 1984
8:00 a.m.
8:15 a.m.
Agenda Item
Individual
10:30 a.m.
11:00 a.m.
11:30 a.m.
12:00 Noon
3. Status of Wastewater Workgroup
4. Status of Nonaqueous Liquid Wastes
Workgroup \
5. Status of Soils and Sediments
Workgroup
6. Status of Waste Solids Workgroup
Adjourn for Day
(a)
Introductory Remarks
Workgroup Meetings
1. List workgroup tasks
2. Continue revising protocols per
group discussion
3. Resolve issues and summarize
Workgroup Summary Presentations
• Summary of workgroup consensus
• Group discussion
• Final recommendations
1. Summary of Air Protocol
2. Summary of Drinking Water Protocol
3. Summary of Wastewater Protocol
Lunch
D. Brusick
Y. Wang
K. Brown
B. Scott
L. Williams
L. Williams
Workgroup
Members
V. Ramanujam
W. Tabor
D. Brusick
continued-
(a) Meeting room available for informal work group meetings in the evening to
achieve consensus on unresolved issues.
225
-------
Appendix 2 - continued
Time
Wednesday
continued
1:30 p.m.
2:00 p.m.
2:30 p.m.
3:00 p.m.
4:40 p.m.
4:50 p.m.
5:00 p.m.
Workgroup Summary Presentations (continued)
4. Summary of Nonaqueous Liquid Wastes
Protocol
\
5. Summary of Soils and Sediments Protocol
6. Summary of Waste Solids Protocol
Preparation of Draft Workgroup Reports and
Revised Protocols
W°rk8r0up
Closing Remarks
Ad j ourn
Individual
Y. Wang
K. Brown
B. Scott
Workgroup
Members
and Revised j. Beaton
L. Williams
226
-------
APPENDIX 3
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY PROTOCOL
REVIEW OF LITERATURE
Introduction
Residue organics isolated from numerous environmental, waste and
biological samples have been tested for mutagenicity and_°?;« "jjj
points via a variety of short-term bioassay procedures (Hoffman 1982).
studies have pursued three lines of investigation: (1) development of
short-term bioassays to assess the mutagenicity/carcinogenicity of the residue
organics; (2) development of isolation methods to prepare samples of residue
organics from environmental, waste and biological matrices that are suitable
for bioassay and (3) development of fractionation methods to isolate
individual mutagens/carcinogens from the complex mixture of residue organics
for further biological assessment or for compound identification. Progress in
the area of short-term tests of individual chemicals and of "Jjtures of
residue organics has been noteworthy over the past decade. This progress has
been documented in numerous publications, including published reports of a
biannual symposium on short-term testing of complex mixtures (Waters et al.
1978 1980, 1983, 1984), of guidelines and recommendations of the use or
short-term tests (e.g., the USEPA Gene-Tox Program and the program of the
International Commission for Protection Against Environmental Mutagens and
Carcinogens, both of which are periodically detailed and updated In the
•Journal, Mutation Research), of guidelines for the assessment of toxicity and
biohazards of environmental mixtures (e.g., Level 1 Biological Testing Program,
USEPA 1981, 1983) and of an interim quality assurance program for the use or
the Salmonella mutagenicity test in the bioassay of chemicals and mixtures of
residue organics (Williams and Preston 1983). However, progress in the area
of sample preparation has been less dramatic. The protocols detailed in this
document address the gaps of standardization and recommendations of procedures
for the isolation of residue organics from environmental and waste samples for
mutagenicity testing. One problem associated with each of these protocols is
the lack of approaches to the fractionation of the complex mixtures of residue
organics. Such separation methods are necessary, not only for the afore-
mentioned reasons, i.e., compound isolation for chemical identification and
further biological testing, but also to address problems associated with the
initial bioassays of the residue organics prepared via the protocols. At
least two problems are apparent: (1) results of the initial bioassay of the
residue organics showing toxicity instead of a dose response and (2) results
of the initial bioassay showing no response. Both of these results could be
due to the presence of antagonistic and/or toxic components present in the
original mixtures of residue organics. Therefore, an initial fractionation of
227
-------
tu ^
tionation procedures have been indicated for "f,' ^ tyI"S °f frac'
-
assessment andfor cornd identmtion ^h*" f?""™1
of these methods, with particular att^?™ , ^ ""J features <•* the use
In the following sections attention to HPLC techniques, are discussed
mixtures of residue ont
the protocols
the complex
and ««• -<«ple. via
ssss Jo
the desired crlterl escrlbed abe
--
add1"011 '° Close1''
these samples
Subsequent studies by Schuetzle et al
HPLC of mutable components of '
228
-------
matter from dlesel exhausts. One important point of this extensive study was
that the HPLC procedure gave >95% recoveries of both the mass and mutagenic
activity of the sample extracts of residue organics. Other noteworthy studies
of the residue organics from air samples via HPLC include: Those of Leary et
al (1983), in which phenalen-1-one was identified as a toxicologically sig-
nificant component in fossil fuel combustion emission; those of Tokiwa et al.
(1983) in which mutagenic nitro compounds were found on exposure of nonmuta-
genic aromatic/heterocyclic constituents of airborne particulates to nitrogen
dioxide; those of Harris et al. (1984), in which nitropyrene was shown to be a
contributing mutagenic factor in coal fly ash particles and those of Alfheim
(Alfheim and Ramdahl 1984, Alfheim et al. 1984), in which wood combustion
emission extracts of residue organics were shown initially to be toxic on
mutagenicity testing, and HPLC fractionation led to a more accurate assessment
of the mutagenic potential of these samples. The conclusion drawn from these
representative studies of residue organics isolated from air samples is that
HPLC can be effectively utilized to separate extracts for a more accurate
determination of the mutagenicity associated with the organic constituents.
Fractionation Studies of Residue Organics Isolated from Drinking Water and
Wastewaters—
Samples of residue organics from both drinking water and the aqueous
portions of wastewater are prepared according to similar protocols, as
described in this document. Therefore, fractionation studies of the residue
organics from both sample types for mutagenicity testing will be considered
together in this section. The solids portions (i.e., sludges) of the waste-
water samples will be discussed in the section on Soils and Sediments.
The need for the fractionation of residue organics isolated from water
samples to assess mutagenicity was presented in an overview of short-term
biological tests by Loper (1980a). The toxicity and nonlinear dose-response
curves obtained on initial bioassy of such residue organics appeared to be
commonplace for these samples. Therefore, sample fractionation is warranted
to more accurately assess the mutagenicity of these samples.
The use of HPLC techniques to separate residue organics from wastewater
samples for further characterization was introduced by Jolley et al. (1975).
Crathorne et al. (1979) proposed an HPLC approach to the identification of
organic constituents in the residue organics of drinking water. At this time,
Jolley and Gumming (1979) separated complex mixtures of nonvolatile organics
in polluted waters by HPLC for bioscreening. A more general HPLC approach was
introduced by Tabor (Tabor et al. 1980, Tabor and Loper 1980) for the frac-
tionation of residue organics from drinking water that featured assessment of
the distribution of residue organics by polarity using an analytical HPLC
separation followed by preparative scale HPLC separation on the residue
organics for mutagenicity testing. This approach led to the first identifi-
cation of a previously unidentified mutagen from drinking water residue
organics (Tabor 1983).
The use of HPLC for the separation of residue organics from aqueous
samples for mutagenicity testing has been extended to the studies of many other
types of water samples from different parts of the world. Baird and co-workers
(Baird et al. 1980, Nellor et al. 1984) have utilized HPLC in their studies of
229
-------
genotoxic components of oil shfle retort dls±nfectlon -thodB. In a study of
first separated these samples by a classicIl^mHJ1^' ^rnlste et al- (1983)
followed by HPLC separation of the e f lcal ll(luld-liquid extraction scheme
group (Kool et al. 1984) has reported th b±oassays. One Dutch research
water residue organics for mutagenicity assessment!1"0 *° fraCt±°nate dr^king
£ ^ '~'i~tt- of drinking »ater
toper et al. 1983, 1984 aor and lo^ ?Jj J J-d.Loper (citations above,
studies, residue organics fZ Tvar^t of ^cer "
=:
.
230
-------
have been reported by Haugen et al. (1982) and Benson et al. (1982) for the
illation of fractions of biohazardous substances in other synthetic fuel
products.
The application of HPLC to the separation of residue organics from solid
fractions for a variety of short-term bioassays.
Fractionation Studies of Residue Organics from Biological Samples for
** jrSoitLS^-Idu. organics isolated from the six media ^c
this document, residue organics have been isolated for a var let y of biologic al
samples and subsequently fractionated via HPLC for mutagenicity testing A
few examples of these studies are warranted. Putzrath and co-workers (Putzratn
et al 1981, Putzrath and Eisenstadt 1983) have reported the isolation of
residue organics from urine for subsequent fractionation via reverse phase
HPLC for the isolation of fractions for mutagenicity testing Similar studies
genicity tests.
Use of HPLC for the Fractionation of Residue Organics
As can be seen, HPLC has been applied successfully to a wide variety of
samples of residue organics for the preparation of residue organics for
mutagenicity testing. A few comments on the use of the technique and possible
limitations are warranted.
ev recommended for the HPLC separation of
residue organics for subsequent mutagenicity testing was based upon a timber
of investigations. These have included applicability to a variety of samples,
choice of columns and mobile phases and subsequent preparation of the HPLC
subtractions for bioassays. As previously noted, HPLC has been applied to a
wide variety of samples of residue organics.
To separate these residue organics, the choice of HPLC columns has been
an outgrowth of the nature of the separation question. The most widely used
type of HPLC column for the separation of residue organics has been the
reverse phase column, i.e., a silica gel modified to an '"'l"*1"^ <"f
Tabor 1984 for a discussion of the types and mechanisms of HPLC separations).
In a reverse phase HPLC separation, the more polar constituents in a sample
are eluted first, followed by the elution of increasingly nonpolar constitu-
ents It is with this latter group of organic compounds, i.e., the nonpolar
constituents, that reverse phase has the widest application to the
231
-------
envi™'
example, Fan et al. (1977) isolate £ SVbfractions for bioassays. For
cutting flulds via .ll^cSSitSr^'"'^" ^^iethanolamine from
used for the fractionation of some ai? Lmt ' IS™11 phaSS HPLC has been
et al. 1984). Other applications of L™?/6^^^83111" (e'8" Alfheim
residue organics for muLgenicity tfstZ I P ^ HPLC tO the sePa«tion of
of the previous sections. testing care are contained in the citations
phase
' normal
. '
and waste sample, the technique is^ot onlv hifh? °r8anlr frOm envir°nmental
but also gives good recoveries of residue La "J 7 fProd«cible and reliable,
mutagenicity (e.g., Schuetzle et al 198? °Tr8atllcs ^ terms °* mass and
Loper 1984b). This is important wL ™ IfT* Bnd Tab°r 1983' Tabor and
mutagenicity of the sample crn^ J balances and accountability of
Such is th/case in types 0^""^^ /" ±m?™a™ criteria for a study.
this document. For example ??C selarltl™* J 6 pr°tocols described in
good recoveries of mutagenic comnon^f ^Ot glVe ^antitative or even
residue organics (see ?fbor et T ?980 JS*"^ r°m/°mPlex ^tures of
separation). Likewise, many liquid-Hcufd TTf ,S,°f S6Veral types of TLC
show evidence of altering not onlv Vh J f , liquid-solid extraction schemes
residue organics but also £e amount of ^ position of the samples of
et al. 1984). From the results of Jh! r^*™8** of the sai»Ple (see Tabor
sections, HPLC appears to b" superior to?LC Lde'l ^ ^ r"^1118
recovery and sample alterations! extraction in terms of
of
the use of HPLC for the separation
^^
alcohols, phenols)
genicity testing. The procedure «
terms of HPLC system compat
should be miscible, and the
-tector (e.g. ,
Proces^d for muta-
the attendant Protocol. In
example, Maron et
—ved from the HPLC
232
-------
with the Salmonella mutagenicity test, whereas Ande rs°!.a!jeM^^1{a
reported that although some solvents are compatible with the Salmonella
muSgenicity test, the use of different solvents with comparable samples gave
difXrent yields of revertants. Therefore, HPLC subtractions should be
prfpSd in comparable ways for mutagenicity testing
solvent removal is via gentle evaporation (e.g . , Ba lrj
MSO) or mutagenesis testing. A description of th J
solutions passed through a sample enrichment and P""*^""
%PP PAK(aM containing octadecylsilane. Following removal of the last
portion of ol'nt from the SEP-PAK the HPLC subfract ion residue organics are
elutad with a small volume of methylene chloride. The eluate is then concen
trated via evaporation for mutagenicity testing. This procedure is detailed
further in the protocol section.
Recommendations and Limitations— „,„„«, ai1hf ructions or
The goal of this fractionation procedure is to prepare subtractions or
residue organics suitable for mutagenicity testing. This is necessitated by
analytical scale reverse phase (or normal phase i.
tlon of an aliquot of the residue organics to assess the dlj5^"°"f°Jhe
organic constituents in the sample according to polarity. The bulk of the
sample is separated via preparative scale HPLC, and the subtractions are
processed for bioassay. The criteria for using reverse phase or normal phase
SH.£lS In the Protocol section. The method has been shown to^vid.l
applicable and to give reproducible, quantitative results without Artifacts o
isolation (citations above) . The method also can be used as a startxng pol nt
for the isolation of mutagens from residue, organics for compound identifi-
cation and chemical/biological characterization.
There are several limitations to the method. For application to mixtures of
residue organics containing salts or ionic substances, the general approach
described in the Protocol is applicable, but columns containing different
(a) Registered trademark.
233
-------
ca^^^ Thi. area needs further
for suitability. A second lim±t«Mn J ^l™*** °n a case-by-caSe basis
serious. The successful us^ of the HPLC°ni \ " TT ^ althOUgh lfc ls n
residue organics will reauire Lmo\ v Protocol for the separation of
S SS
..
to the user of thH ^otocol! artiCle8 °n H?LC that
234
-------
REFERENCES
Alfheim I, Becher G, Hongslo JK, Ramadhl T. 1984. Mutagenicity testing of
high performance liquid chromatography fractions from wood stove emission
samples using a modified Salmonella assay requiring smaller samples. Environ.
Mutagen. 6:91-102. ^
Alfheim I, Ramdahl T. 1984. Contribution of wood combustion to indoor air
pollution as measured by mutagenicity in Salmonella and polycyclic aromatic
hydrocarbon concentration. Environ. Mutagen. 6:121-130.
Anderson D, McGregor DB. 1980. The effect of solvents upon the yield of
revertants in the Salmonella/activation mutagenicity assay. Carcinog.
1:363-366.
Baird R, Gute J, Jacks C, et al. 1980. Health effects of water reuse: A
combination of toxicological and chemical methods for assessment. In: Jolley
RL, Brungs WA, Gumming RB, Jacobs VA, eds. Water chlorination: Environmental
impact and health effects, Vol. 3. Ann Arbor, MI: Ann Arbor Science
Publishers Inc., pp. 925-935.
Benson JM, Mitchell CE, Royer RE, Clark CR, Carpenter RL, Newton GJ. 1982.
Mutagenicity of potential effluents from an experimental low BTU coal
gasifier. Arch. Environ. Contam. Toxicol. 11:547-551.
Cheh AM, Skochdopole J, Heilig C, Koski PM, Cole L. 1980. Destruction of
direct-acting mutagens in drinking water by nucleophiles: implications for
mutagen identification and mutagen elimination from drinking water. In:
Jolley RL, Brungs WA, Gumming RB, Jacobs VA, eds. Watet chlorination:
Environmental impact and health effects, Vol. 3. Ann Arbor, MI: Ann Arbor
Science Publishers Inc., pp. 803-815.
Claxton LD. 1983. Characterization of automotive emissions by bacterial
mutagenesis bioassay: a review. Environ. Mutagen. 5:609-631.
Crathorne B, Watts CD, Fielding M. 1979. Analysis of non-volatile organic
compounds in water by high-performance liquid chromatography. J. Chromatogr.
185:671-690.
Cumming RB, Lee, NE, Lewis LR, Thompson JE, Jolley RL. 1980. Relationship
of disinfection to mutagenicity in wastewater effluents. In: Jolley RL,
Brungs WA, Cumming RB, Jacobs VA, eds. Water chlorination: Environmental
impact and health effects, Vol. 3. Ann Arbor, MI: Ann Arbor Science
Publishers Inc., pp. 881-898.
235
-------
««•««
«»««.
2?582-M7. ' °e P88e '"""""on. M«u*I Chromatography and HPLC
M°5rl!°n J> »«™"1>«U« DP, Ross R, Pine DH, Miles W 1977
B,
s?
-iron.
236
-------
Jolley RL, Gumming RB. 1979. Oridatit effects on complex mixtures of non-
volatile organics in polluted waters: examination by HPLC and bioscreening.
Ozone Sci. Eng. 1:31-37.
Jones AR, Guerin MR, Clark CR. 1977. Preparative scale liquid chromato-
graphic fractionation of crude oils derived from coal and shale. Anal. Chem.
49:17661771.
Kool HJ, van Kreijl CP. Verlaan-de Vries M. 1984.
tion and characterization of organic mutagens in drinking water
IH, Malaiyandi M, eds. Concentration techniques for collection and analysis
of organic chemicals for biological testing of environmental samples. Washing
ton, DC: American Chemical Society (in press).
Leary JA, Lafleur AL, Liber HL, Biemann K. 1983. Chemical and
characterization of fossil fuel combustion product phenalen-1-one. Anal Chem.
55:758-761.
Loper JC. 1980a. Overview of the use of short-term biological tests in the
assessment of the health effects of water chlorination. In: Jolley RL,
Brungs WA, Gumming RB, Jacobs VA, eds. Water chlorination: Environmental
impact and health effects, Vol. 3. Ann Arbor, MI: Ann Arbor Science
Publishers Inc., pp. 937-945.
Loper JC. 1980b. Mutagenic effects of organic compounds in drinking water.
Mutat. Res. 76:241-168.
Loper JC, Tabor MW. 1983. Isolation of mutagens from drinking ™ter;
something old, something new. In: Waters MD, Sandhu SS, Lewtas J, Claxton L
Chernoff N, Nesnow S. Short-term bioassays in the analysis of complex environ-
mental mixtures III. New York: Plenum Press, pp. 165-181.
Loper JC, Tabor MW, Miles SK. 1983. Mutagenic subtractions from nonvolatile
organics of drinking water. In: Jolley RL, Brungs WA, Cotruvo JA, Cummxng
RB! Mattice JS, Jacobs VA, eds. Water chlorination: Environmental impact and
health effects, Vol. 4, Book 2, Ann Arbor, MI: Ann Arbor Science Publishers
Inc., pp. 1199-1210.
Loper JC, Tabor MW, Rosenblum L, DeMarco J. 1984. Continuous removal of both
mutagens and mutagen forming potential by a full scale granular activated
carbon treatment system. Environ. Sci. Technol. (in press).
Maron D, Katzenellenbogen J, Ames BN. 1981. Compatibility of organic solvents
with the Salmonella/microsome test. Mutat. Res. 88:343-350.
Marshall MV, Noyola AJ, Rogers WR. 1983. Analysis of urinary mutagens
produced by cigarette-smoking baboons. Mutat. Res. 118:241-256.
Mutation Research, Amsterdam, The Netherlands: Elsevier Biomedical Press.
237
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sanitation
Saltation Ditricts o,c'o reP°rt' """""' CA=
testing of
L, Chernoff N, NesnoTVedsT Shoit-t.™'!^' ^^J" ^' LeWtaS J' Claxton>
-i . ' cu°«» onorc—term bioasfiavs ^n «-h« „„_-! j_ ^*?
Res. 85:97-108. ^ -gn-perrormance liquid chromatography. Mutat
\
, Rubin IB, Guerin MR, Clark BR. 1981.
__ f?—9fl o
bioassay of crude synthetic fuels? "Mutat.
in . o£ Seph LB ""
'81' e -lf of
238
-------
Tabor MW, Loper JC. 1984a. Analytical isolation, separation, and identifi-
cation of mutagens from nonvolatile organics of drinking water. Int. J.
Environ. Anal. Chem. (in press).
Tabor MW, Loper JC. 1984b. Mutagen isolation methods: fractionation of
nonvolatile residue organics from aqueous environmental samples. In: Suftet
IH, Malaiyandi M, eds. Concentration techniques for collection and analysis
of organic chemicals for biological testing of environmental samples. Washing-
ton, DC: American Chemical Society (in press).
Tabor MW, Loper JC, Myers BL, Rosenblum L. Daniels FB. 1984. I^ion of
mutagenic compounds from sludges and wastewaters. Inv: Waters MD, Sandhu SS,
Hueisingh JL, Claxton L, eds. Short-term genetic bioassays in the evaluation
of complex environmental mixtures, IV (in press) New York, NY: Plenum Press.
Tokiwa H, Kitamori S, Nakagawa R, Ounishi Y. 1983. Mutagens in airborne
particulate pollutants and nitro derivatives produced by exposure of aromatic
compounds to gaseous pollutants. In: Waters MD, Sandhu SS, Lewtas J, Claxton
L, Chernoff N, Nesnow S. Short-term bioassays in the analysis of complex
environmental mixtures III. New York: Plenum Press, pp. 555-567.
Toste AP, Sklarew DS, Pelroy RD. 1982. Partition chromatography-high per-
formance liquid chromatography facilitates the organic analysis and biotesting
of synfuels. J. Chromatogr. 249:267-282.
USEPA 1981 U. S. Environmental Protection Agency. Office of Research and
Development. IERL-RTP. Procedures manual: level 1 environmental assessment
billogLal tests. Research Triangle Park, NC: U. S. Environmental Protection
Agency. USEPA-600/8-81-024.
USEPA 1983. U. S. Environmental Protection Agency. Office of Research and
Development. IERL-RTP. Quality control and quality assurance procedures for
level 1 health effects bioassays. Research Triangle Park, NC: U. S. Environ-
mental Protection Agency. USEPA-IERL-RTP-S1463.
U. 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.
Waters MD, Sandhu SS, Huisingh JL, et al., eds. 1978. Application of short-
term bioassays in the fractionation and analysis of complex environmental
mixtures. New York: Plenum Press.
Waters MD, Sandhu SS, Huisingh JL, et al., eds. 1980. Short-term bioassays
in the analysis of complex environmental mixtures II. New York: Plenum
Press.
Waters MD, Sandhu SS, Lewtas J, et al., eds. 1983. Short-term bioassays in
the analysis of complex environmental mixtures III. New York: Plenum Press.
239
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240
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PROTOCOL FOR THE SEPARATION OF RESIDUE ORGANICS FOR MUTAGENICITY TESTING
1.0 Scope and Application
1 1 This method is to be used for the separation of residue organics
Isolated from environmental matrices for subsequent mutagenesis bioassay of
the separated organics.
1 2 The method is suggested for residue organics that show toxicity or no
dose response on initial mutagenesis bioassay. the method is applicableto
residue organics isolated from environmental and waste samples via methods
described in this protocol document.
1.3 The method can be used as a starting point for the isolation of mutagens
from residue organics for compound identification and chemical/
biological characterization.
1 4 The method is applicable to mixtures of residue organics containing
ioitlyslipolar and nonpolar constituents. For application to mixtures of
residue organics containing salts or ionic substances, the general approach is
applicable! but different chromatographic stationary phases may be required.
Further research on a case-by-case basis may be required.
1 5 This method is restricted to use by or under the supervision of analysts
experiencedin chromatography and properly trained in the handling and use of
biohazardous materials.
2.0 Summary of Methods
2 1 Residue organics showing toxicity or no dose response on initial muta-
genesis assessment are fractionated via HPLC, and the isolated «*f«ctionB
fre tested for mutagenicity. The separation process involves a minimum of two
steps: an initial analytical scale HPLC separation of an aliquot of the
sample to assess the distribution of constituents in the sample according to
the?r polarity and a preparative scale HPLC separation of the sample, wherein
fractions are collected for mutagenesis bioassay.
2 2 The method described in this protocol is based on reports by ^°* and
Umer (1980, 1984a, b), Tabor et al. (1980, 1984), Loper and Tabor (1983),
ioper it .?. (1983 1984), Baird et al. (1980), Nellor et al, (1984) and
Alfheim et al. (1984) for the fractionation of complex matures of residue
organics isolated from environmental and waste samples. The design and
application of the method are described in the above list of references. The
method has been successfully used in the separation of residue organics
241
-------
p ,
the original mixtu^of ^i^T^s ° "* -'M-i"* constituants of
3-0 Definitions
defines Ty™ ^itlJS*^?""",*0"*' f°r TeStl"8 Materials (ASTM)
*
,
electrica conductivity at 25*0 oH 0^ ^ "*"** °f °'1 ^/L- •
resistivity at 25 C of 16 67 Mnh °-06/mho/^> • -Inlau. electrical
potassium permanganate of 60 v^'™ *** * ^^^ COl°r retentlon tl« for
3.2 HPLC - High Performance Liquid Chromatography
L'tal
tal anspls viaotocol HCS' S°ted from envi
nesis testing. protocols described in this document for mutage-
solvents,
residue organics.
4.0 Interferences
5.0 Safety
T Carpinno>er^r<-t»•« «* ___jj
tn this
- » ———» "t"upj.e and subsequent HPLC
treated as a potential health hazard.
have been described (USNCI 1981).
6>0 Apparatus and Materials
6.1 HPLC Unit—
242
-------
generating combinations of isocratic and gradient solvent elutlonj during
I ample separation (Waters Associates Model 680 or equivalent), a ^posi-
tion automatic solvent switching valve (Waters Associates Autochrome 101 or
equivalent), and a variable volume, to 2.0 mL, loop injector (Waters
Associated Model U6K or equivalent). All connecting tubing should be of
stainless steel and TFE composition.
612 A radial compression model HPLC column unit (Waters Associates
RCM! 00(l) Number 84800 or Waters Associates Z-Module(a> Number 86500 or
equivalent) fitted with 8 mm by 10 cm Radial-PAKU; cartridge packed with 10 u
oc?adecyfsilane (Waters Associates number 84770 or equivalent) for reverse
phase separation or packed with 10 u silica (Waters Associates number 84730 or
equivalent) for normal phase separations. Alternatively, other types of pre-
parative scale HPLC columns, reverse phase and normal phase, may be ^ed,
provided that these columns are capable of separating at least 75 mg of resi-
dueorganics with the resolution (Tabor 1984) obtainable on an analytical
scale, i.e., 25 yg of sample.
6.1.3 A mercury lamp UV detector fitted with a 254 nm filter (Waters
Associates Model 440 or equivalent).
6 1 4 Two pen recorder for monitoring both UV detector output and solvent
composition fisher Scientific Recordall^ Series 5000, Number 13-939-20 or
equivalent).
6.1.5 Sample evaporator (Organomation Associates, Inc., N-Evap a Model 111
or equivalent).
6.1.6 Evaporative concentrator, modified micro Synder, 4 mL tube (Kontes
No. K-569250 or equivalent).
6.1.7 Analytical balance, readable to 0.01 mg with a precision of ± 0.01 mg.
6.1.8 Muffle furnace, capable of sustaining 500 C, for use in glassware
decontamination.
6 1 9 Solvent clarification kit (Waters Associates Number 85113 or
equivalent) for use with HAWPO 4700 aqueous filters (Waters Associates
Number 85117 or equivalent) and FHUP 04700 organic filters (Waters Associates
Number 85118 or equivalent).
6.1.10 Sample enrichment and purification cartridges, packed with
octadecylsilane (Waters Associates SEP-PAK Number 51910 or equivalent).
6 1 11 Guard column (Waters Associates Number 84550 or equivalent) packed
with octadecylsilane (Waters Associates Number 27248 or equivalent) for
reverse phase separations or with silica (Waters Associates Number 27245 or
equivalent) for normal phase separations.
6 1 12 Sonication - A sonicator capable of holding two or more 1-L solvent
containers for solvent degassing (Branson Model B-32 or equivalent).
243
-------
6.2 Cleaning of Apparatus and Glassware—
AS™
6.2.4 General glassware - clean as recommended in 44 PR 69464, December 3,
.7.0 Reagents
stord ln •* ^- or e,ulva-
8'° HPLC Separation of Residue Organics
8.1 Solvent and Sample Preparation—
~
HPLC us, has ,n descrid ^*S!2 0984)
: •;:«, Sfjs.-sfss.vs
244
-------
volume of methylene chloride. By adding hexanet^ solution is diluted using
the same method described for acetonitrile solutions.
8 2 Analytical Scale HPLC Separations—
.'.,., Hearse «piTSS! s£ r.i.
.
phase mode, in order to characterize the sample of r on of
——
.ith vater for 5 min or unti no more ^. ^^
eluted .
of this HPLC run, the system is re-equilibrated to
After
before injecting the next sample.
EeBultS fro. thl. HPLC separation
sample of the residue organic, J^^^'nitrne'or iatr, then proceed
at a solvent composition of 80% wa"'-™ "' tlon of the sample to
to the preparative scale reverse phase HPLC separat ion o r ^ ^
sstr— - ""
of the residue organics is conducted if t be "J" s sPaccompiished by injecting
not suitable, as described above ^ ^"^ciinto^he HPLC unit operat-
25 yL of a 1 Pg per yL solution of "•1J^0*^XC;.J ^/min; initial mobile
ing under the following correlat ions ^rbance u^ts full scale. Following
int cos for10 o until no more
Ssorbing, i.e. peaks >5% full scale, components are eluted.
245
-------
the
methanol to 100% tnethylene chloride- a
10 min linear gradient from 1007 m
wash of the column, with W/hLln
results of this HPLC separation will
residue organics, in terms
8.3 Preparative Scale HPLC Separations—
' ^1
to
.8'8dle« from 100%
Wlth ^thylene chloride; a
Chloride to 100% hexane; a 15 min
*1"8 *"? "^ Sample' The
con's tit ^nt's'^ °f ^ "^ °f
the
complex mixture oresue
are applicable to 40 to 80
2.0 absorbance
full
until no
the collection of Fraction
peaks
below,
the eluate is collected as rrac
to a new one for the collection
*"* SePa^ion, one of
be ut"i"d for the
**
detCtOr Settln8 of
„
srr ;:
are eluted;
- 3
no nore ro
i-adlately, as descrlbed
collection vessel is changed
246
-------
more UV absorbing (peaUs >5% full scale ) component, a eluted •
time, the eluate continues to be collected as Action B. Fra
S'T^-^^^S = -
Fractlon c,
nsitsr^s^r:i^t5?Si «: r
collection of Fraction C continues during the wash.
.
„
=a
absorbing (i.e., >5% full scale), components are eluted.
The seven Fractions, A-G, are processed itmnediately as described
below, for bioassay.
protocols in this document, and each is described below.
organics
247
-------
cols of
according to
--•-'"'""=' j-ium tin; manuractiifpf« <-HQ CTPD DAI/ • *_i . e
of Type I water. Following the slow n^* SEP-pf " then washed with 20 mL
the SEP-PAK -5 ml nf .^J8 f passage of the HPLC subtraction through
in Teflon-capped amber vials at -20 J'nnM? ?*' ^ theSG Sai"pleS are Stored
solutions are then nrnZ^ " J° ^Until muta8e"esls testing. These
Section 8.4.1
9.0 Blanks and Controls
9.1 Solvent Blanks —
9.2 HPLC System Blanks—
^^
nol. blle Phase 8radle"t from methylene chloride to metha-
248
-------
if an analytical solvent blank run does not meet specifications. If the
second cleaning procedure does not restore the column performance adequately,
the HPLC column is replaced.
9 2 2 Preparative scale HPLC blanks— Following a preparative scale HPLC run,
natal or reverse phase, the appropriate analytical scale solvent blank run is
performed. If the column needs cleaning, the procedure in Section 9.2.1 is
followed.
10.0 Sample Storage
Acetone and methylene chloride concentrates of the HPLC subtractions
of residue organics are stored in Teflon-capped amber vials, containing an
inert gas (nitrogen or helium) atmosphere, at -20 C until mutagenesis testing
or further chemical analysis/HPLC separation. 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 un-
published) .
11.0 Data Records
Records to be maintained on the sample include a description of the
residue organics sample to be separated, information on the preparation of the
residue or^anics, mutagenesis data for the initial testing of these samples
and chain of custody forms for each sample. Additionally, records to be
maintained on the HPLC separation include HPLC system performance data,
chromatograms on both analytical and preparative fPa™tion*»/°^tf H?LC
subtractions collected, data on quality control of columns and S°^^S,
volumes of concentrated HPLC subtraction extracts and storage data and any
unusual occurrences during the HPLC separations. Chain of custody forme
should be executed for each sample from the HPLC. procedure through the muta
genesis testing step.
Records needed to be maintained for the columns include each lot of
SEP-PAK cartridges, each lot of solvents used and the overall maintanence/
system performance of the HPLC system.
12.0 Calculations
From the data records, the final volume and/or weight of each HPLC
subtraction is related directly back to the sample of residue organics and
also to the original environmental or waste sample used in the preparation of
the residue organics. Therefore, it is imperative that accurate records of
all weights/volumes be maintained throughout all operations.
249
-------
REFERENCES
Alfheim I, Becher G, Hongslo JK, Ramadhl T. 1984. Mutagenicity testing of
s±3P TanCG ^"J" chr^tography fractions from wood stove emission
Mutagen.U6:9?-l02? Salm°nella assa? re«uiri"g «-"er samples. Environ.
Dilution' fmdahl ^ v1984* Contrlbutlon of wood combustion to indoor air
hvdiocarL meaSUred by ""tagenicity in Salmonella and polycyclic aromatic
hydrocarbon concentration. Environ. Mutagen. 6:121-130.
Anderson D, McGregor DB. 1980. The effect of solvents upon the yield of
"I6e- " thS ^lm°nella/aCtivation ""tagenicity assay. "
tion'of 'tOKicolo , na
chemical methods for assessment. In: Jolley RL,
'
Sr f f Mltf el1 CE' R°yer RE, Clark CR, Carpenter RL, Newton GJ. 1982.
Mutagenicity of potential effluents from an experimental low BTU coal
gasitier. Arch. Environ. Contain. Toxicol. 11:547-551.
Cheh AM, Skochdopole J, Heilig C, Koski PM, Cole L. 1980. Destruction of
direct-acting mutagens in drinking water by nucleophiles: implications for
mutagen identification and mutagen elimination from drinking water In:
Jolley RL, Brungs WA, Gumming RB, Jacobs VA, eds. Water chlorination:
SM^nmnaJ ^PaCT ^ health effects» Vol. 3. Ann Arbor, MI: Ann Arbor
Science Publishers Inc., pp. 803-815.
Claxton LD. 1983. Characterization of automotive emissions by bacterial
mutagenesis bioassay: a review. Environ. Mutagen. 5:609-631.
Crathorne B, Watts CD, Fielding M. 1979. Analysis of non-volatile organic
chromatography. J. Chromatogr.
Cumming RB, Lee, NE, Lewis LR, Thompson JE, Jolley RL. 1980. Relationship
of disinfection to mutagenicity in wastewater effluents. In: Jolley RL
11 A KUIT^8 «' JaC°bS VA' eds' Water ^lorination: Environmentll
3- Ann Arb- MI:
250
-------
Dion P, Bruce WE. 1983. Mutagenicity of different fractions of extracts of
human feces. Mutat. Res. 119:151-160.
Dolan JW. 1984. Mobile phase preparation. Liquid Chromatography and HPLC
2:582-587.
Salmonella/microsome test. Cancer Res. 38:431-438.
term bioassays in the analysis of complex environmental mixtures III.
York: Plenum Press, pp. 59-78.
Eisenberg WC 1978. Fractionation of organic material extracted from "
suspended air particulate matter using high pressure liquid chromatography. J.
Chromatogr. Sci. 16:145-151.
1980. The use of short-term tests in the isolationjmd
mutagens in
Mutagens 6.
impurity. Science 196:70-71.
Goeckner HA, Griest WH. 1977. Determination of methyl chrysenes in a coal
liquefaction product. Sci. Total Environ. 8:187-193.
^^^«•-
^
6:131-144.
=^^^r^-r^
Anal. Chem. 54:32-37.
Hoffman GR. 1982. Mutagenicity testing in environmental toxicology.
Environ. Sci. Technol. 16(10) :560A-572A.
Jolley RL, Katz S, Mrochek JE. 1975. Analyzing organic in complex, dilute
aqueous solutions. Chem. Technol. 5(5) : 312-318.
251
-------
Jolley RL, Gumming RB. 1979 Or-M*«* fe
nonvolatile organics in polluted ™? '" °n C°mplex ml*tures of
ing. Ozone Sci. Eng. l~:?l-37 W3terS: exami*ation by HPLC and bioscreen-
Jones AR, Guerin MR, Clark CR. 1977 Pr^a>- +<
SS^I^Jr*1" °f C-de °"° ^"Pa^^1Ve •«1--^" chroma-
n o crue c±s d -
Chem. 49:17661771. S derived from coal and shale. Anal.
»~L^
, e « wtr h >
and health effects, Vol. 4 Boik 2 I!n J K "i10"'' Env±ronm^tal impact
Inc., pp. 1199-1210. 2' Ann Arb°r' MI: Ann Arbor Science Publishers
f 1JJ4. Continuous removal of both
carbon treatment system. ™ "
-:
. .
Mutation Research, Amsterdam, The Netherlands: Elsevier Biomedical Press.
252
-------
Sanitation Districts of Los Angeles County.
n» ™ ^ says *- «^~
environmental mixtures III. New fork: Plenum Press, pp. 79-87
Res. 85:97-108.
bioassay of crude synthetic fuels. Mutat. Res. 85.29-39.
Reddy BS, Sharrna C, Mathews L, Engle A. 1984 Fecal mutagens fro, subjects
consuming a mixed-western diet. Mutat. Res. 135:11-19.
"
Singh I. Lusby AF, McGuire PM. 1982. Mutagenicity of HPLC fractions from
extracts of AAtex-treated corn. Environ. Mutagen. 4:45-53.
environmental mixtures III. New York: Plenum Press, pp. 139-151.
8:197-215.
Tabor MW, Loper JC, Barone K. 1980. Analytical procedures for fractionating
mutagenic components from drinking water concentrates, n.
Science Publishers Inc., pp. 899-912.
Tabor MW. 1983. Structure elucidation of 3-(2-chloroethoxy)-l,2-Dichloro-
propene, a new promutagen from an old drinking water residue. Environ. Sci.
Technol. 17(6) : 324-328.
Tabor MW. 1984. Chromatography: theory an d practi ce In: Ka plan LA Pesce
AJ, eds. Clinical Chemistry: theory, analysis and correlation. St. Louis,
-
XlxJ J *_-vi.tj • « .— .— ..— — — — — - ~
MO: The C.V. Mosby Co., CH 4, pp. 74-99.
253
-------
* *' Analytlcal isolation, separation, and
organics °f
Mutagen lsolatlon methods: fractionation of
Tabor MW, Loper JC, Myers BL, Rosenblmn L, Daniels FB. 1984 Isolation
' S"d8 ^ •»"» "
Tokiwa H, Kitamori S, Nakaeawa R Oin'«?M v io«rj M ,.
,
biotesting of synfuels. J. Chromatogr. 249:267-282.
USEPA. 1981. U. S. Environmental Protection Agency. Office of Research and
~ "
Protection
USEPA. 1983. U. S. Environmental Protection Agency. Office of Research and
'11311^ C°ntr01 and ^al±t? ASS— *ro"£r« ?or
l athff, SS— ror« or
Level 1 Health Effects Bioassays. Research Triangle Park, NC- U S Environ
mental Protection Agency. USEPA-IERL-RTP-S1463. r » Wl" u- ^ Environ
InStltute' 1981' Office of Research Safety. The safe
? sr -1- - the
mixtures. New York: Plenum Press.
thee!Lf)'-SandhU SS,' Hulsln8h J0' et -I- eds.. 1980. Short-term bioassays in
the analysis of complex environmental mixtures II. New York: Plenum Press.
theeana^^anfU ^ ^^ J> et 3l" 6dS' 1983' Sh°«-term bioassays in
the analysis of complex environmental mixtures III. New York: Plenum Press.
theean«f ''SandhU ^ LeWtaS J' Gt 3l" eds' 1984' Short-term bioassays in
(in press)"8 ** envlr°nmental -*«ures IV. New York: Plenum Press?
254
-------
Williams LR, Preston JE. 1983. Interim ^oced;re" -o
Saltnonella/Microsomal Mutagenicity Assay (Ames Test). EPA-600/4-82-068,
Environmental Monitoring Systems Laboratory, Las Vegas, HV.
:" #U.S. GOVERNMENT PRINTING OFFICE-^ 55 9-W 3/20020
255
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