5773
IAG No. DOE-IAG-40-646-77
EPA-IAG-78-D-X0372
TOXICITY OF LEACHATES
INTERIM PROGRESS REPORT
APRIL 1, 1978 TO JANUARY 1, 1979
J. L. Epler, Principal Investigator
W. H. Griest, Analytical Chemistry
M. R. Guerin, Analytical Chemistry
M. P. Maskarinec, Analytical Chemistry
D. A. Brown, Environmental Sciences
N. T. Edwards, Environmental Sciences
C. W. Gehrs, Environmental Sciences
B. R. Parkhurst, Environmental Sciences
B. M. Ross-Todd, Environmental Sciences
D. S. Shriner, Environmental Sciences
H. W. Wilson, Environmental Sciences
F. W. Larimer, Biology
T. K. Rao, Biology
Oak Ridge National Laboratory
Oak Ridge, Tennessee 37830
Operated by
Union Carbide Corporation
for the
U.S. Department of Energy
Prepared for the
Office of Solid Waste
U.S. Environmental Protection Agency
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IAG No. DOE-IAG-40-646-77
EPA-IAG-78-D-X0372
TOXICITY OF LEACHATES
INTERIM PROGRESS REPORT
APRIL 1, 1978 TO JANUARY 1, 1979
J. L. Epler, Principal Investigator
W. H. Griest, Analytical Chemistry
M. R. Guerin, Analytical Chemistry
M. P. Maskarinec, Analytical Chemistry
D. A. Brown, Environmental Sciences
N. T. Edwards, Environmental Sciences
C. W. Gehrs, Environmental Sciences
B. R. Parkhurst, Environmental Sciences
B. M. Ross-Todd, Environmental Sciences
D. S. Shriner, Environmental Sciences
H. W. Wilson, Environmental Sciences
F. W. Larimer, Biology
T. K. Rao, Biology
Oak Ridge National Laboratory
Oak Ridge, Tennessee 37830
Operated by
Union Carbide Corporation
for the
U.S. Department of Energy
Prepared for the
Office of Solid Waste
U.S. Environmental Protection Agency
-------
m
CONTENTS
1 . Executive Summary 1
2. Introduction 7
2.1 . Background 7
2.2. Scope of Work 7
3. Sampling 9
3.1 . Sample Handling 9
3.2. Sample Description 9
3.3. Preliminary Sample Data 12
4. Extraction Procedure 15
4.1 . Problem Definition 15
4.2. Extracting Apparatus 15
4.3. Results and Discussion 17
5. Chemistry 21
5.1 . Problem Definition 21
5.2. Scope of Work 21
5.3. Results 24
5.4. Discussion 33
5.5. Conclusions 38
6. Aquatic Toxicity 39
6.1 . Problem Definition 39
6.2. Scope of Work 39
6.3. Results 40
6.4. Discussion 43
6.5. Conclusions 46
7. Mutagenicity 49
7.1 . Problem Definition 49
7.2. Results 50
7.3. Discussion 51
7.4. Conclusions 64
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IV
8. Phytotoxicity 67
8.1 . Problem Definition 67
8.2. Methodology 67
8.3. Results 68
8.4. Discussion and Conclusions 72
9. References 75
10. Appendices 77
I. Extraction Procedure 77
II. Extractor 79
III. Analytical Methodology 91
IV. Aquatic Toxicity Methodology 95
V. Salmonella Mutagenicity Assay 97
VI. Saccharomyces cerevisiae Gene Mutation Assay 109
VII. Bacterial DMA Repair Assay 113
VIII. Seed Germination/Radicle Length Assay 117
IX. Seedling Growth Assay 121
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ABBREVIATIONS
AAS atomic absorption spectrometry
ANPR Advance Notice of Proposed Rulemaking
Ar Aroclor
As arsenic
ASTM American Society for Testing Materials
BAP benzo(a)pyrene
DMSO dimethylsulfoxide
EP Extraction Procedure
EPA Environmental Protection Agency
GC gas chromatography
ORNL Oak Ridge National Laboratory
PAH polyaromatic hydrocarbon
PCB polychlorinoted biphenyl
0B phenobarbital
RCRA Resource Conservation and Recovery Act
SLT Standard Leaching Test
VOA Volatile Organics Analysis
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1. EXECUTIVE SUMMARY
Under Subtitle C of the Resource Conservation and Recovery Act (RCRA) of
1976 (PL 94-580), the Environmental Protection Agency (EPA) is required to pro-
mulgate regulations for the management of hazardous waste. To assist the EPA in
developing characteristics for identifying wastes which, due to their toxic nature,
pose a potential hazard to human health and the environment, the Oak Ridge
National Laboratory (ORNL) has conducted studies on (1) leaching of toxicants
from waste materials, (2) analytical procedures for characterizing extracts of
wastes, and (3) screening bioassays for evaluating the toxicity of extracts. This
report summarizes work during the period April 1, 1978 through January 1, 1979.
Experimental work during this reporting period has concentrated on:
(1) extracting four wastes (fly ash, scrubber sludge, soybean process cake,*
and bottom ash) by use of the procedure proposed in the Federal Register on
December 18, 1978 (43 FR 58956) in order to uncover potential problem areas;
(2) evaluating various analytical methodologies selected during ORNL's
earlier work to determine their suitability for analyzing wastes and waste extracts;
(3) evaluating methodologies for preparing concentrates of the organic
materials present in the extracts suitable for mutagenicity testing;
(4) evaluating short-term in vitro mutagenicity bioassays;
(5) evaluating screening assays for toxicity to aquatic organisms and
terrestrial plants;
(6) determining the utility of these procedures by use of the extracts prepared
from the four wastes and from a sample of groundwater contaminated with arsenic.
The soybean process cake was evaluated because earlier work indicated that waste
from edible oil processing may be mutagenic.
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Extraction Procedure (EP). Extractors of the type described in Figure 1 of
43 FR 58961 were used to perform the extractions on the wastes. They were
constructed of polymethylmethacrylate resin and fabricated by the Environmental
Engineering Department of the University of Tennessee. However, while these
extractors satisfactorily agitated the wastes, there was occasional jamming during
extraction of bottom ash. The extractor was redesigned to improve the alignment
of the rotor and container so as to minimize jamming, lessen the possibility of
abrasive damage to the unit during extraction of hard materials, and allow the use
of organic solvent containing waste materials. This new extractor was similar to
the previous unit. By changing the materials of construction to Type 316 stainless
steel, improving the bearing design, lessening manufacturing tolerances, and
redesigning the container and motor mount, we were able to increase both the
versatility of the extractor and the operator productivity.
A study was also conducted to compare atomic absorption spectrometry (AAS)
with other analytical methods for the measurement of metals in the EP extracts.
(Wherever the "EP extract" is specified, this is actually a combination of an acidic
aqueous extract of the solid portion of the waste and the original liquid present in
the sample.) The AAS methods used were those in the then current EPA Standard
Methods for Water and Waste (EPA-62516-74-003), except that to increase sensi-
tivity a graphite furnace was used in place of the conventional flame. Analytical
techniques included spark source mass spectrometry, inductively coupled plasma
emission spectrometry, optical emission spectroscopy, and neutron activation analysis.
The results indicate that AAS compares well with other methods for metal analysis.
The uncertainty of the analytical procedures for the heavy metals of interest ranged
from 1 to 10 percent in the concentration range of concern (0.1 to 10 mg/liter).
When these wastes were evaluated, the groundwater contaminated with
arsenic (As) was found to be the only one which exceeded the threshold for a toxic
waste as defined in 43 FR 58956 because it contained both 41 2 mg/liter of arsenic
and 0.49 mg/liter of cadmium. The only other waste which might be labeled toxic
by these criteria would be the fly ash by virtue of its borderline extract cadmium
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level (0.1 mg/liter). However, taking into account the blank (0.01 mg/liter)
would drop the level to marginally below the threshold.
Aquatic Toxicity. The objective of the aquatic screening tests was to
determine the toxicity and potential hazard of waste extracts to aquatic biota.
The tests used were acute (short-term lethality) tests and chronic (long-term) tests
designed to measure sublethal effects on reproduction. The test organism was the
cladoceran Daphnia magna, an aquatic organism sensitive to most classes of toxic
chemicals.
The As-contaminated groundwater exhibited toxic effects in both acute and
chronic aquatic bioassays; however, based on the threshold for a toxic waste
described in the Advance Notice of Proposed Rulemaking (ANPR) 43 FR 59022 it
would not be ranked as a toxic waste. On the other hand, the soybean process
waste exhibited neither acute nor chronic toxic effects in the test employed. The
three power plant wastes (fly ash, flue gas desulfurization sludge and bottom ash)
showed only marginal toxicity, well below the aforementioned threshold levels.
The 28-day chronic toxicity test appears to show the presence of toxic
materials in the As-contaminated groundwater. Daphnia reproduction, however,
is greatly influenced by the food supply, and the extracts often contain high con-
centrations of acetate. The acetate may be a substrate for the bacteria, which are,
in turn, fed on by the daphnids, and which in turn would result in an increase in
number of young produced. For the controls in which acetic acid was used, it was
found that the production of young was in fact significantly higher than when
dilution water only was used.
Acute toxicity tests (48-hr, LCj-n) were conducted on extracts before and
after storage at 4 C for 28 days. Because of the low toxicities of the extracts,
reliable 90 percent fiducial limits could not be obtained. However, the data
indicated no statistically significant change in toxicity.
Mutagenicity. The mutagenicity protocol is intended to serve as an
indicator of the chronic hazards of mutagenicity and carcinogen!city. Because of
systemic differences in reactivity to mutagenic substances, a battery of assays has
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been employed by ORNL with eukaryotic and prokaryotic organisms to detect both
point mutation specifically and DMA damage generally. The vast majority of
known chemical mutagens are organic in nature. Because the concept of threshold
is ill-defined for genetic activity, and because the EP extract of solids was antici-
pated to be low in organic character, it was deemed advisable to examine a
concentrated extract of the organic constituents of the EP extract in addition to the
extract itself.
Mutagenicity as defined in the proposed regulations 43 FR 58965 (for
delisting) and the AN PR 43 FR 59023 requires the application of three assays for
determination of whether or not a waste is a hazardous waste: (1) gene (point)
mutations in bacteria; (2) gene mutations in eukaryotes, either in mammalian
somatic cells in culture or in fungal microorganisms; (3) recombinogenic or repair-
related phenomena. The tests used in the ORNL program for each category were.-
(1) the Salmonella/microsome assay, (2) the Saccharomyces can/his dual assay,
and (3) the Salmonella uvrB repair assay.
Arsenic-contaminated groundwater, four EP extracts, and XAD-2 concen-
trates of all five samples were tested in each assay both with and without metabolic
activation. Both Aroclor- (Ar) and phenobarbital- (0B) induced rat liver S-9 mix
were used in the studies with metabolic activation. The XAD-2 concentrates of the
EP extracts achieved a 250-fold (v/v) concentration of the organic material, while,
because of sample limitations, only a 12.5-fold concentration could be obtained on
the contaminated groundwater.
None of the tested samples displayed toxicity in the Salmonella/microsome
assay in the dose ranges tested, and only in the case of the As-contaminated ground-
water was mutagenic activity found. It was slightly mutagenic with the frameshift
strain TA98, but only upon mutagenic activation with Ar-induced S-9. The XAD-2
concentrate, however, was mutagenic with the frameshift strains TA1537, TA98, and
TA100. It did not require metabolic activation and, in fact, the addition of the
S-9 mix (either Ar- or 0B-induced) reduced the mutagenic activity.
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In the Sacchciromyces mutation assay the As-contaminated groundwater
sample was not mutagenic. However, the XAD-2 concentrate was mutagenic
without metabolic activation after a 24-hr exposure. Metabolic activation
appeared to reduce the mutagenic potential of the concentrate. None of the
power plant or soybean process wastes or concentrates exhibited mutagenic activity
either with or without activation. In addition, none of the test materials appeared
to be toxic to the test organism.
The As-contaminated groundwater exhibited moderate toxicity to the
organisms in the DNA repair assay. However, none of the test samples displayed
mutagenic activity either with or without S-9 activation.
Of the five wastes discussed in this report, only the As-contaminated
groundwater possessed detectable mutagenic activity. For purposes of bioassay,
the mutagenic principal in the undiluted waste water is at the limit of resolution,
the XAD-2 concentrate being necessary to conclusive demonstration of mutagenic
activity. Work is in progress to determine if the o-nitroaniline known to be in
the waste accounts for the mutagenic properties.
The response of the Salmonella/microsome assay to the As waste implies a
frameshift mutation mechanism which requires the addition or deletion of DNA
base-pairs. This is supported by the yeast results with this waste, which show a
moderate preponderance of induced forward mutation to can , relative to induced
reversion of the his base-pair substitution. This is a typical response (by this
system) to a frameshifting agent.
The As-contaminated groundwater and its XAD-2 concentrate failed to
elicit a response from the bacterial DNA repair assay. There are two key consider-
ations: (1) the overall mutagenic potency of the waste is moderate, (2) validation
studies have shown that the repair assay is particularly insensitive (although not
unreactive) to frameshifting agents. Hence, it may not be significant to obtain a
negative result in this context.
With regard to the negative results obtained with the power plant wastes and
the soybean process cake, the aqueous extracts and XAD-2 concentrates of these
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materials are extremely deficient in organic character. The majority of organic
mutagens are not detectable at the parts-per-billion level. Furthermore, inorganic
mutagens (e.g., metals and metal complexes) which might be present are commonly
not detectable by the bioassays in question.
Phytotoxicity. One objective of this project was to develop a short-term
assay for screening wastes for phytotoxicity. Two tests were studied: a 48- and
72-hr root (radicle) elongation test for radish and sorghum, respectively, and a
2-week seedling growth study for wheat and soybean. Test seeds or plants were
exposed to various concentrations of the EP extracts or As-contaminoted groundwater
to determine their toxicity, the ultimate objective being to see if a 10 percent
concentration of the extract is significantly toxic to the plants. This concentration
is that which the EPA scenario indicates could be used for irrigation. The As-
contaminated groundwater was found to be highly phytotoxic, causing a 33 percent
growth reduction in the radish roots even at a 2 percent concentration; however,
at a 0.1 percent concentration there was a slight stimulation of root growth. None
of the waste extracts showed toxic effects in all tests. The scrubber sludge was toxic
to radish seeds in the root elongation test even at a 10 percent concentration, but the
same concentration was not toxic to sorghum. Fly ash, soybean process cake, and
bottom ash were only slightly toxic to radish and sorghum at concentrations exceeding
10 percent. In the seedling growth studies, the fly ash and soybean wastes caused
a slight reduction of root weight but not shoot weight.
In both the short- and long-term tests with the soybean process cake, the
dicotyledons (radish and soybeans) were not affected, whereas the roots of the mono-
cotyledons (wheat and sorghum) were reduced significantly.
One significant potential problem relates to the presence of high concentra-
tions of acetate in extracts of highly basic wastes. Early results confirm literature
reports that acetate is phytotoxic. While this did not cause difficulties in the
samples evaluated during this period, work is under way to further define the magnitude
of the potential problem.
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2. INTRODUCTION
2.1 . Background
Under the terms of the RCRA of 1976 (PL 94-580), EPA is required to pro-
mulgate regulations for the management of hazardous waste. Section 3001 of
Subtitle C of RCRA further requires EPA to develop and promulgate criteria for
identifying the characteristics of hazardous waste and for listing particular wastes
which are subject to the regulations. To assist the EPA in developing characteristics
for identifying wastes which, due to their toxic nature, pose a potential hazard to
human health and the environment, ORNL entered into an interagency agreement
>
with EPA in 1977. Under the terms of this agreement ORNL would conduct studies
to evaluate leaching and bioassay tests, recommend toxicity test protocols, and
perform analytical, biological, and environmental sciences testing to establish a
preliminary toxicity data base on leachates.
The work is a cooperative effort between ORNL's Biology, Environmental
Sciences, and Analytical Chemistry Divisions.
2.2. Scope of Work
Samples of various wastes have been provided to ORNL by the Hazardous
Waste Management Division, Office of Solid Waste, EPA; the American Society for
Testing Materials (ASTM); and other groups.
Based on the needs of EPA's Office of Solid Waste, emphasis during this work
period has been on:
(1) evaluating the EP proposed in the Federal Register on December 18,
1978 (43 FR 58956) to measure the tendency of toxicants to migrate from a waste
under "improper" disposal conditions,
(2) evaluating the following short-term, inexpensive screening tests to
determine whether the introduction of such extracts to the environment might pose a
substantial present or potential hazard to human health or the environment: (1) 48-hr
D. magna LCc/y (2) 28-day D. magna reproduction; (3) Salmonella/microsome
bacterial reversion; (4) Saccharomyces mutation; (5) Salmonella-uvrB repair;
(6) Radish, wheat, and sorghum radicle length; (7) Wheat and soybean seedling
growth.
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8
Experimental work during this reporting period has concentrated on;
(1) testing a series of wastes using the EP;
(2) evaluating various analytical methodologies selected during ORNL's
earlier work to determine their suitability for analyzing waste extracts (with
emphasis on identifying and determining the concentration in the extracts of those
species on the National Interim Primary Drinking Water Standards list and the
Priority Pollutants list);
(3) evaluating methodologies for preparing concentrates of the organic
materials present in the extracts suitable for mutagenicity testing;
(4) developing and evaluating short-term in vitro mutagenicity bioassays;
(5) developing and evaluating screening assays for toxicity to aquatic
organisms and terrestrial plants;
(6) determining the utility of these procedures by use of extracts prepared
from various wastes.
Earlier work under this contract leading up to the current EP will be published as
part of the final project report scheduled for early fall of 1979.
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9
3. SAMPLING
3.1 . Sample Handling
Figure 3-1 shows the stepwise disposition of a given waste as it proceeds
through analysis at ORNL. The Environmental Sciences Division (ESD) executes
the EP on the waste and distributes the resulting EP extract to the Environmental
Sciences Division for phytotoxicity and aquatic screening assays and to the
Analytical Chemistry Division (ACD) for further processing. The Analytical
Chemistry Division performs inorganic and volatile organic analyses on the EP
extract; prepares XAD-2 resin concentrates of the EP extract suitable for poly-
nuclear aromatic hydrocarbons, polychlorinoted biphenyls, and pesticide analyses
following chemical fractionation; and provides aliquots of the EP extract and its
XAD-2 resin concentrate to the Biology Division for mutagenicity bioassay.
3.2. Sample Description
The following materials, covering wastes expected to be both hazardous
and nonhazardous, were supplied by EPA or ASTM for use in this project: (1) As-
contaminated groundwater, (2) organoarsenical waste, (3) metal cleaning waste,
(4) dyestuff manufacturing sludge, (5) latex carpetbacking sludge, (6) plating sludge,
(7) soybean process charcoal filtration cake; (8) raw shale, (9) shale retort waste,
(10) fludized bed combustion waste, (11) municipal sewage sludge, (12) coal-fired
power plant fly ash, (13) coal-fired power plant bottom ash, (14) flue gas desulfuri-
zation sludge, (15) API separator sludge.
This report describes the work conducted on the extraction and testing of the
following materials: (1) As-contaminated groundwater, (2) fly ash, (3) scrubber
sludge, (4) bottom ash, (5) soybean process charcoal filtration cake.
Four of the wastes were extracted by use of the EP described in the proposed
regulations (Appendix I). A previous publication described the philosophical
basis of the EP. Three of the wastes represent different solid coal combustion residues.
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10
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11
These wastes were fly ash, scrubber sludge, and bottom ash. The fourth waste,
soybean process cake, is a solid disposed of after the bleaching of corn syrup; it was
2
selected for use in this program as a result of the observation during an earlier study
that an edible oil processing waste showed mutagenic activity. The As-contam-
inated groundwater sample was distributed for testing as received, since it was a
liquid and did not have to be extracted by the EP. Detailed information regarding
the chemical and physical characteristics of the wastes was not provided with the
samples. The results from studies conducted on the remaining samples will be re-
ported in the final project report.
All waste samples received for testing were placed outdoors in an open shed.
This was a safety precaution deemed necessary due to the lack of information
concerning the nature of the samples. The representativeness of the waste sample
is not assumed, since the waste was stored for an extended period of time and other
collection and storage factors were unknown, such as the procedures used to collect
the samples (i.e., ORNL did not know whether the sampling protocols cited in the
proposed hazardous waste regulations were used), temperature changes prior to
arrival at ORNL, and possible sample-container interactions. It is unreasonable
to assume or predict changes (biological, chemical, or physical) that may occur in
a sample over time. These topics, though, are a necessary consideration in the
final version of the hazardous waste characterization procedure. Therefore, it
should be noted that the waste testing being conducted was to assess the adequacy
of the testing protocols cited in the hazardous waste regulations, rather than to
determine whether the wastes were hazardous.
Additionally, a standard procedure was not applicable in obtaining a
representative subsample of the wastes for extraction. The types of wastes to be
tested ranged from a dry powder to sludge and slurry. Therefore, upon examination
of each waste, a judgment was made as to how an aliquot of the sample would be
taken. In most cases thorough mixing or quartering was utilized.
It is our recommendation that more specific sampling guidelines be incorp-
orated into the EP itself, rather than into the Appendix of the regulations. The
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12
draft report, Handbook for Sampling Hazardous Waste (Research Grant
R-804692010), seems to be an adequate reference source for this information.
3.3. Preliminary Sample Data
Preliminary laboratory data collected by ORNL on the waste samples
included:
(1) Solids content, calculated as the weight in grams of the sample after
drying divided by the original weight in grams of the sample.
(2) General physical description including color, odor, and consistency.
(3) Preliminary titration of a 5.0-g sample with 0.5 N acetic acid.
A 1:16 solidrsolution ratio was used to give an indication of the amount
of acid required for pH adjustment during the sample extraction. Table 3-1
summarizes these data for the four waste samples. While these wastes were of
varying moisture content, ranging from a low of 1 percent moisture to a high of
59 percent, they were all what would normally be considered to be solids. One
area worthy of comment concerns the need for care to be taken during all phases of
the sampling and testing phases to prevent loss of sample moisture. This is espe-
cially important for high moisture content wastes. A change in moisture content
due to evaporation of water from the sample from 90 percent to 80 percent would
change the contribution of the original liquid phase to the final EP extract from
31 percent to only 20 percent. Thus it is very important to ensure that the sample
tested is truly representative of the waste sampled.
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15
4. EXTRACTION PROCEDURE
4.1 . Problem Definition
Prior to initiation of this study, EPA had commissioned a study by Dr. R.
Ham and his co-workers at the University of Wisconsin to develop a Standard
Leaching Test (SLT). The purpose of this test was to devise a procedure which
would model waste disposal in a landfill in admixture with a decomposable organic
waste such as refuse.
Specifically EPA was interested in determining what components of the waste
would be likely to migrate out of the waste under such a disposal environment. It
was the intent of ORNL and EPA that the ORNL work would be geared toward
developing methodology to measure whether the leachate posed a potential hazard
to either man or the environment.
However, soon after initiation of the work at ORNL, it was learned that
while the SLT did a good job of modeling landfill conditions, the inherent toxicity
of the SLT extraction fluid was such as to preclude determination of the additional
toxicity caused by material migrating from the waste and thus make SLT unsuitable
for the planned use of bioassay procedures at some later date. Thus the ORNL
work program was modified by EPA to include testing of a second-generation EPA
leaching method and to determine if it was compatible with bioassay procedures for
determining the toxicity of the waste. It should be noted that EPA's intent in
developing the EP was (1) to "model" improper management based on wastes creating
a problem through migration of chemicals out of a disposal site, and (2) to use this
with the resultant analysis as a screen to separate out those wastes which required
special handling. The contamination scenario has been developed for definition
purposes only; it is not meant to address actual site specific disposal methods which
might be used for a particular waste.
4.2. Extracting Apparatus
Extractions were performed with extractors of the type described in 43 FR
58961 at the direction of the Project Officer. To save time, prototype extractors
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16
constructed of Plexiglas were borrowed from the Environmental Engineering Depart-
ment of the University of Tennessee. However, due to the possible problems of
abrasive damage during extraction of hard materials, the solubility of the Plexiglas
in organic media present in some wastes, and the possibility of jamming (due to
misalignment), an improved version of the extraction equipment was designed and
fabricated at ORNL from Type 316 stainless steel. Details of materials and design
can be found in Appendix II (Figures II-l through II-l 1).
As shown in Appendix II, the most important improvements of the new design
are the use of Type 316 stainless steel in all parts which are exposed to the waste or
extract, as requested by EPA, and the unitizing of the vessel, supporting frame,
and stirring motor. Type 316 stainless steel, a high quality steel, was considered
an improvement because of its inherent strength and resistance to abrasion, a factor
which is especially important when dealing with hard granular wastes such as bottom
ash. The integration of the vessel, support stand, stirring rod, and stirring motor
is seen to be the best way of assuring positive alignment of the agitator blade and
vessel bottom. This feature minimizes the probability of waste particles binding
and scraping between them and assures a smooth mixing action. A high-torque,
low-rpm stirring motor and its accompanying solid-state controller have proven more
than adequate for the job and provide positive, variable-speed propulsion of the
stirring rod. The conical bearing surface for the stirring rod on the bottom of the
vessel is an important improvement in that it assures positive centering of the stirring
rod in the vessel and minimizes grinding action and clogging between the stirring rod
end and the vessel bottom. This unitized system allows close tolerances to be
maintained between the edges of the stirring blade and the bottom and sides of the
extraction vessel, thereby assuring thorough mixing of the solid and liquid phases
during the EP.
One potential problem remains to be resolved regarding the possible leaching
of metals from the walls of the vessel and stirring rod. The extent of this problem is
being verified at this time. A possible alternative is to have the vessel and stirrer
fabricated out of Tefzel (a DuPont registered trademark).
-------
17
Due to the lack of additional specification in the EP, the laboratory
practices and utilization of reagents cited in Appendix II was deemed more than
adequate. We recommend the specification of water and chemical grades as an
addition to the EP.
4.3. Results and Discussion
Data recorded during the extraction of the four wastes are represented in
Table 4-1. The following parameters were recorded for each individual extraction;
initial pH, final pH, amount of 0.5 N acetic acid initially added to adjust the
solution to pH 5, total 0.5 N acetic acid added during the 24-hr extraction period,
and electrical conductivity of the final extract. The solution pH was automatically
adjusted during all extractions.
Following extraction, the solid and liquid phases were again separated by use
of the Millipore filtration system. A 0.45-u.m pore size filter was utilized for final
filtration. In all cases filtration was accomplished by means of a vacuum filter
which proved to be satisfactory for all wastes extracted to date. It should be noted
that the EP extract is a combination of the original liquid from the waste sample and
the liquid resulting from the extraction.
Data recorded upon completion of the extractions are given in Table 4-2.
The following parameters were recorded for the extract in its final form: pH,
electrical conductivity, total 0.5 N acetic acid added, color, and remarks on
unusual appearance or occurrence.
Upon completion of the EP, aliquots of the extract were delivered to the
appropriate groups at ORNL for analysis. When delivery could not be accomplished
immediately after completion of the procedure, all extract samples were refrigerated
at 4C.
We recommend that standardized procedures for storing the EP extract prior
to analysis be incorporated into the final protocol.
-------
18
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TABLE 4-2
EXTRACT DATA
Extract
Fly ash
Scrubber
sludge
Bottom
ash
Electrical
pH conductivity
(jj mho/cm)
4.8 1910
4. 9 2340
5.0 154
Total acetic
acid added
(mEq/g sample
extracted)
0.191
0.429
0.026
Color
transparent, colorless
transparent, colorless
(white precipitate formed
after addition of super-
natant to leachate the
precipitate was filtered
out, and both were sent
separately to Analytical
Chemistry Division)
transparent, colorless
Soybean 5.2
process
cake
240
0.077
transparent, yellow
-------
21
5. CHEMISTRY
5.1. Problem Definition
Due to the need of the Office of Solid Waste for rapid, reliable analytical
methods for the analysis of the 14 substances (i.e., eight heavy metals and six
pesticides) for which threshold levels have been proposed (Table 5-1), the
Analytical Chemistry Division of ORNL has been concerned with the validation
of analytical techniques and quantitative analysis of the extracts obtained from the
EP. In addition, ORNL has also been concerned with the validation of analytical
techniques for the concentration extraction, and quantitative analysis of the
Priority Pollutants (Table 5-2) in extracts obtained from the EP. The Priority
Pollutants fall into several distinct chemical classes: polyaromatic hydrocarbons
(PAH's), phenols, pesticides and polychlorinated biphenals (PCB's), volatile halo-
organics, phthalates, various nitro and halo-substituted benzenes and toluenes.
The objective of this work has been to identify and measure in a cost-effective
manner as many of these compounds as possible at one-tenth the level indicated in
Table 5-1 or 1 mg/liter for those species listed in Table 5-2.
In addition to characterization and measurement, an additional aspect has
been to develop methodology for concentrating the organic constituents of the
extract in a manner suitable for testing in the short-term bioassays described in the
section on mutagenicity testing.
5.2. Scope of Work
Many instrumental techniques are available for the measurement of metals in
a liquid waste or EP extract. A study was conducted to compare the commonly used
AAS with other techniques. The AAS methods used were those specified in the
current EPA Standard Methods for Water and Wastes (as specified in the proposed
regulations) except that a graphite furnace was employed instead of the conventional
flame. This change has since been incorporated in the Standard Methods. Ana-
lytical techniques examined for analyzing extracts include spark source mass
-------
22
spectrometry (including isotope dilution where applicable), inductively coupled
plasma emission spectrometry, optical emission spectroscopy, and neutron activation
analysis. A collaborative AAS analysis of an EP extract also was carried out with
the Illinois State EPA, and two EPA Laboratory Performance Evaluation Standards
supplied by the EPA Environmental Monitoring and Support Laboratory (Cincinnati)
were used to test the accuracy of our AAS methodology.
Preparation of Organic Concentrates. Our initial attempts to assay the
aqueous EP extracts directly were unsuccessful from the standpoints of both organic
analytical characterization and mutagenicity bioassay because of the low concen-
trations of the extracted constituents. Thus, we conducted an investigation of
available methods for concentrating the organics present in the extracts. Methods
for separating the extracts into chemically simpler fractions and for analyzing
volatile organic compounds and metals also were examined.
TABLE 5-1
PROPOSED EP EXTRACT THRESHOLDS FOR DESIGNATING
A WASTE AS A TOXIC WASTE
Substance
Ag
As
Ba
Cd
Cr
Hg
Pb
Se
2,4-D
Lindane
Methoxychlor
Toxaphene
2,4,5-TPSilvex
Endrin
Threshold level (mg/liter)
0.5
0.5
10.0
0.1
0.5
0.02
0.5
0.1
1.0
0.04
1.0
0.05
0.1
0.002
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23
TABLE 5-2
EPA PRIORITY POLLUTANT LIST
Acenaphthene
Acrolein
Acrylonitrile
Aldrin/Dieldrin
Antimony and compounds
Arsenic and compounds
Asbestos
Benzene
Benzidine
Beryllium and compounds
Cadmium and compounds
Carbon tetrachloride
Chlordane (technical mixture and
metabolites)
Chlorinated benzenes (other than
dichlorobenzenes)
Chlorinated ethanes (including 1,2-
dichloroethane, 1, 1, 1-trichloro-
ethane, and hexachloroethane)
Chloroalkyl ethers (chloromethyl,
chloroethyl, and mixed ethers)
Chlorinated naphthalene
Chlorinated phenols (other than those
listed elsewhere; includes trichloro-
phenols and chlorinated cresols)
Chloroform
2-Chlorophenol
Chromium and compounds
Copper and compounds
Cyanides
DDT and metabolites
Dichlorobenzenes (1,2-, 1,3-, and 1,4-
dichlorobenzene)
Dichlorobenzidine
Dichloroethylenes (1, 1-and 1, 2-dichloro-
ethylene)
2, 4-Dimethylphenol
Dinitrotoluene
Diphenylhydrazine
Endosulfan and metabolites
Endrin and metabolites
Ethylbenzene
Fluoranthene
Haloethers (other than those listed
elsewhere; includes chlorophenyl-
phenyl ethers, bromophenylphenyl
ether, bis(dischloroisopropyl) ether,
bis(chloroethoxy) methane, and
polychlorinated diphenyl ethers)
Ha Iomethanes (other than those listed
elsewhere; includes methylene
chloride, methylchloride, methyl-
bromide, bromoform, dichlorobromo-
methane, trichlorofluoromethane,
trichlorodifluoromethane)
Heptachlor and metabolites
Hexachlorobutadiene
Hexachlorocyclohexane (all isomers)
Hexachlorocyclopentadiene
Isophorone
Lead and compounds
Mercury and compounds
Naphthalene
Nickel and compounds
Nitrobenzene
Nitrophenols (including 2,4-dinitro-
phenol, dinitrocresol)
Nitrosamines
Pentachlorophenol
Phenol
Phthalate esters
Polychlorinated biphenyls (PCB's)
Polynuclear aromatic hydrocarbons
(including benzanthracenes, benzo-
pyrenes, benzofluoranthene,
chrysenes, dibenzanthracenes, and
indenopyrenes)
Selenium and compounds
Silver and compounds
2,3, 7, 8-Tetrachlorodibenzo-p-dioxin
(TCDD)
Tetrachloroethylene
Thallium and compounds
Toluene
Toxaphene
Trichloroethylene
Vinyl chloride
Zinc and compounds
-------
24
Solvent partitioning by use of cyclohexane and dichloromethane, and
macroreticular resin extraction with XAD-2 (refs. 3,4) were investigated as
methods for preparing organic concentrates. While determining that the resin
method offered the most promise as a cost-effective method, we realized that the
pH and ionic strength of the EP extract had to be controlled if reproducible con-
centrates were to be obtained.
Fractionation and Analysis of Organic Concentrates. Because the expected.
complex nature of the organic concentrates would seriously limit our ability to
directly identify and measure the various classes of toxicants, we required a means
of separating the organic concentrate into simpler, more easily analyzed fractions.
A modified version of a fractionation scheme developed and validated in this
laboratory for isolation of polyaromatic hydrocarbons from environmental
materials was the most cost-effective approach, and we examined it for its ability
to separate the various classes of toxicants prior to their analysis by gas-liquid
chromatography.
Analysis of Volatile Organic Compounds. Because volatile organic com-
pounds are not readily determined in the organic concentration/fractionation method,
they must be determined by a separate method. The advantages of the purge and
trap method over solvent extraction and other methods for the determination of
volatile organic compounds in water have been well established. We evaluated
a manual version of this method for its applicability to EP extract analysis.
5.3. Results
Table 5-3 is a complete listing of all the EPA Priority Pollutant and Interim
Drinking Water Standard constituents which are specifically determined by this
chemical protocol. The application of this protocol to the EP extracts and ground-
water sample is discussed below.
-------
25
TABLE 5-3
SPECIFIC COMPOUNDS SELECTED FOR MEASUREMENT IN EP EXTRACTS
AND AS-CONTAMINATED GROUNDWATER SAMPLE
PAH*
Acenapthene
Fluoranthene
Naphthalene
1, 2- Benz(a) anthracene
Benzo(a)pyrene (BAP)
Chrysene
Anthracene
1, ]2-Benzoperylene
Fluorene
Phenanthrene
Dibenz(a,c and a,f)anthrenes
Pyrene
Volatile
1, 1-Dichloroethylene
Methylene chloride
trans- 1,2-Dichloro-
ethylene
Acrolein
Dichlorobromomethane
Tetrachloroethylene
Bromoform
Bis-[2-chloroethyl]
ether
Acrylonitrile
1, 1-Dichloroethane
Chloroform
1,2-Dichloroethane
Trichloroethylene
2-Chloroethyl vinyl
ether
1, 1,2-Trichloroethane
Chlorodibromomethane
S-Tetrachloroethane
Element
Ag
As
Ba
Be
Cd
Cr
Cu
Hg
Ni
Pb
Sb
Se
Tl
Zn
F
PCB/pesticide
PCB (Ar 1242)
Lindane y-
isomer
Methoxychlor
Endrin
Toxaphene
"Plus 31 other PAH's not included on the Priority Pollutant List.
Analysis for Metals. Because of the availability and widespread use of
AAS, it was compared with five other instrumental methods of metals analysis by
assaying an EP extract of sewage sludge. Analyses were conducted in quadruplicate.
The results shown in Table 5-4 indicate that AAS compares well with other methods
of metals analysis. Further evidence for the accuracy of our AAS method was
obtained through a cooperative study arranged with Mr. Frank Schmidt of the Illinois
State EPA. The results of quadruplicate analyses of a sewage sludge EP extract are
-------
26
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27
shown In Table 5-5. Four metals are footnoted as being determined in a second
study; except for these four, the results of the two laboratories in the first study
were in good agreement. The data reported for the second study also agree well,
which implies that a contamination problem (probably incurred during packaging the
EP extract for shipment) caused the disparity of results for these four metals in the
first study.
Finally, two EPA Laboratory Performance Evaluation Standards were analyzed
for their content of As, Be, Cd, Cr, Cu, Hg, Ni, Pb, and Se. According to Dr.
D. E. Banning, the EPA Project Officer, the Be results by AAS were biased slightly
TABLE 5-5
COMPARISON OF ORNL AND ILLINOIS STATE EPA ANALYSES
OF SEWAGE SLUDGE EP EXTRACT
Average concentration ± S.D. (mg/liter)
Metal in study by
A *
Ag
As*
Ba*
Be
Cd
Cr
Cu
Hg*'*
Ni
Pb
Zn
ORNL
0. 0003
0.007 ±0.0002
<0.5
0.0004 ±0.00002
1.2± 0
0.03 ±0.0008
0.7± 0.01
0.027
3.4±0.2
0.03± 0.003
36. 7 ±0.7
Illinois State EPA
<0.005
0.007
0.4
<0.005
1. 1 ± 0
0.04 ±0.005
0.8±0.05
<0.03
4.0 ±0
0.04±0.001
48. 5± 1.7
Result of second study.
Single measurement of Hg.
-------
28
low, but were sufficiently accurate and precise for this study. Thus, we felt that
AAS was validated as an accurate and precise method of metals analysis, and we
adopted it for our standard method.
Preparation of Organic Concentrates. Experiments were conducted to
compare the efficiency of XAD-2 resin with that of solvent extraction (with both
cyclohexane and methylene chloride) in their ability to extract and concentrate
organic contaminants present in an EP extract. For the spiking experiments, a
synthetic EP extract was prepared from 3.4 ml of glacial acetic acid/liter of
triple-distilled water. This "extract" was then spiked with 6x10 to 12 x 10
decompositions/min/liter of the C-labeled organic compounds listed in Table 5-6,
the pH was adjusted to 6.8 with Na,,PO ,, and the conductance was adjusted to
20 mmho/cm with NaCl. These samples were then extracted with five 100 ml
portions of organic solvent or passed through 4 g of XAD-2 by the method [after
ref. 3] described in Appendix III. Our results are shown in Table 5-6. Methylene
chloride was the most efficient solvent for recovering the organics. However, its
use in mutagenic bioassay is questionable. It should also be noted that while the
solvent extractions provide reliable extraction for most of the organics, the XAD-2
also provides a direct 100-fold concentration. Furthermore, as many as 15 EP
extracts may be extracted simultaneously by XAD-2 if a simple peristaltic pump is
used, eliminal-ing considerable technician time. XAD-2 has also been employed
4
successfully elsewhere for isolating mutagens for short-term bioassay. Thus the
XAD-2 method appeared to be the most cost-effective approach to the preparation
of organic concentrates for analysis or mutagenic bioassay and was adopted as our
routine concentration method.
Fractionation and Analysis of Organic Concentrates. The most cost-effective
means of fractionating the organic concentrates into simpler, more readily analyzed
fractions was felt to be a modification of our existing method for isolation of PAH's
from environmental materials. The modified fractionation scheme is shown in
Figure 5-1, .along with the actual fractionation recoveries achieved with the cyclo-
hexane extracts from the organic concentration studies. Specific details of the
-------
29
TABLE 5-6
EFFICIENCIES OF SOLVENT EXTRACTION AND XAD-2 RESIN FOR
CONCENTRATION OF ORGANICS FROM A MOCK EP EXTRACT
Compound
PCB (Ar 1254)
BAP
Naphthalene
Hexadecane
Indole
Phenol
Stearic acid
Stearyl alcohol
Succinic acid
Sitosterol
Cyciohexane
(%)
~100
-100
~100
41
3
~100
96
0
MOO
Methylene
chloride*
(%)
~100
-100
-100
-100
95
99
99
95
0
-100
XAD-2t
(%)
95
82
-100
93
84
70
91
90
0
91
Extraction efficiency only.
Actual recovery after adsorption/desorption and concentration to 5.0 ml.
-------
30
500 ml acetic acid blank
(pH 6.8, conductance 20 mi Hi mhos)
extract 5 X 1 00 cyclohexane
J
cyclohexane extract
94% Stearyl alcohol
41% Indole
100% Hexadecane
82% Sitosterol
100% BAP
100% PCB's
100% Naprhalene
10 g Florisil
extracted water
100% Phenol
100% Succinic acid
31% Indole
6% Stearyl alcohol
4% Sitosterol
150ml 6:1 hex/kenz
11% Indole
8% Stearyl alcohol
78% Hexadecane
80% Napthalene
86% BAP
100% PCB's
150 ml acetone wash of Florisil
64% Stearyl alcohol
20% Indole
6% Hexadecane
50% Sitosterol
14% BAP
20 g alumina
150 ml hexane
46% Hexadecane
30% Napthalene
96% PCB's
2% Stearyl alcohol
150ml 6:1 hex/benz
2% Stearyl alcohol
6% BAP
150ml 2:1 hex/benz
77% BAP
150 ml acetone
11% Indole
4% Stearyl alcohol
2% BAP
Figure 5-1 . Recoveries of tracers in fractionation procedure.
-------
31
fractionation procedure are included in Appendix III. Our results with tracer com-
pounds indicate that nonpolar materials from the EP extracts are separated from polar
materials by adsorption column chromatography on Florisil. The nonpolar organics
then are separated on an alumina column onto a PCB pesticide mono- and diaromatic
fraction, a polyaromatic fraction, and a heteroaromatic fraction. Each fraction is
analyzed by gas chromatography (GC) at the conditions noted in Appendix III.
Recoveries of PAH's from environmental materials are virtually quantitative. PCB
recoveries are being evaluated with Standard Solutions supplied by the EPA
(Cincinnati).
The groundwater sample was known to be contaminated with o-nitroaniline.
Because the XAD-2 procedure is not particularly efficient for concentration of
ionized organics, a separate solvent extraction procedure was employed (Appendix
III) to extract and concentrate the o-nitroaniline for GC analysis. Four methylene
chloride extractions were sufficient to remove all solvent-extractable color from the
groundwater sample.
Analysis of Volatile Organics. The apparatus we employed in the purge and
trap collection of volatile organics is shown in Figure 5-2. The entire collection
and analysis procedure was evaluated with a standard made up in ethylene glycol
with the following toxicants at the 1 mg/ml level: 1,1-dichloroethane; 1,2-
dichloroethane; hexachloroethane; trichloroethane; tetrachloroethane; 2-chloro-
ethyI vinyl ether; chloroform; 1,1-dichloroethylene; 1,2-trans-dichloroethylene;
1 ,2-dichloropropane; 1 ,3-dichloropropylene; (bis)-2-chloroisopropyl ether; bromo-
form; dichlorobromoethane; trichloroethylene; and dichloromethane. Because of
the high volatilities of these toxicants, aliquots of the standard were added to a mock
EP extract only immediately prior to purging. Thermal desorption and GC analysis
of the trapped standards indicated an average precision of ±20 percent. While the
absolute recovery of each toxicant is unknown, recoveries should be highly reproduc-
ible for standards and samples treated in the same manner. Sensitivity is approxi-
mately 0.1 mg/liter when a 1 ,0-ml sample is analyzed. Greater sensitivity could
-------
32
Teflon Stnpppr .
ORNL DWG 79-13149
Tenax Capsule
Condenser
0-Ring with
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z
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Figure 5-2. Purge and trap apparatus.
-------
33
be obtained by use of a larger sample. Further evaluation of this method with a
EPA Evaluation Standard is in progress.
5.4. Discussion
Inorganic Analyses. The results of the inorganic analyses are presented in
Table 5-7. The uncertainty of the analyses at this concentration level typically
ranges from ±1 to -tlO percent. In combination with the organic analyses, the
results seem to indicate that the main constituents of the EP extracts are ionic in
character, as would be expected from the use of a dilute acidic aqueous extractant
fluid. On the basis of 10 times the EPA Primary Drinking Water Standard as the
maximum permissible concentrations (Table 5-1) of dissolved metals in the EP
extracts, the As-contaminated groundwater sample would be labeled toxic from its
content of As and Cd. Considering the unknown history of this sample, our result
for As of 412 mg/liter compares well with the value (480 mg/liter) labeled on the
original sample container, apparently from an analysis conducted elsewhere in
June, 1978. Handling and storage history prior to our receipt of the sample are
unknown, and may contribute to this difference.
Of the remaining four EP extracts, the only one which may be labeled toxic
by these criteria is fly ash, by virtue of its borderline Cd level. The proposed
definition of a hazardous waste does not include a threshold for fluorine. However,
fluoride in the fly ash EP extract was five times the Primary Drinking Water Standard,
or one-half a "10 times standard" level.
It is interesting to note the increasing concentration trend in the EP extracts
of bottom ash < scrubber sludge < fly ash for several of the elements. This obser-
g
vation is consistent with the preferential accumulation of many of these elements in
fly ash vs. bottom ash.
Metals analyses also were performed on the EP extract blanks, as shown in
Table 5-7. All of the metals in the blanks were far below their maximum permissible
concentrations, except for Ba and Zn, which were significant in comparison with the
levels in some of the EP extracts. Thus although concentrations in these blanks were
-------
34
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35
low relative to RCRA thresholds, they still could influence a borderline case, as
for example, the fly ash EP extract.
An optical emission spectroscopic survey analysis was also performed on a
precipitate which formed in the EP extract of scrubber sludge shortly after genera-
tion. As might be expected from the limestone employed in the scrubber, the
major element in the precipitate was Ca. Approximately 0.1 mg/g of Mg and B
also were detected.
Organic Analyses. The first five samples, consisting of one arsenic-
contaminated groundwater sample and extracts from four wastes, were subjected to
the analytical protocols detailed in Appendix III and discussed above. In addition
to these wastes, four EP extract blanks were taken through the same analytical
scheme to evaluate the contribution of the blank to the composition of the EP
extracts. Detection limits for a 500-ml sample were: for PAH's, 2 u.g/liter; for
volatiles, 0.01 ng/liter; for PCB's, 0.1 ng/liter; for pesticides, 0.1-1 ng/liter.
No PAH's, volatiles, or pesticides were detected in groundwater or any of the
extracts or blanks. PCB's were detected at a concentration of ~0.2 ng/liter for the
As-contaminated groundwater and all four extracts; they were detected at a concen-
tration of ~1 ng/liter for all four blanks. The identification of PCB's in EP extracts
is tentative.
For the analysis of purgeable volatiles, a few mi Mi liters of EP extract were
taken immediately after generation, bottled, and refrigerated in order to preserve
any volatiles present in the fresh extract. No volatiles could be detected in these
samples above the 0.1 ng/liter level. Even when the extracts were frozen immedi-
ately after generation, no volatiles were present. This result is not suprising in
view of the fact that the wastes were stirred vigorously in open containers for 24 hr,
so any volatiles present probably were removed to the atmosphere during the EP. To
obtain meaningful data on the content of the volatile organic species in wastes it
will be necessary to analyze the wastes directly. An alternative would be to modify
the EP apparatus so as to continuously purge the system through a Tenax adsorbent
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36
trap during the waste extraction. In any case, the presence of high levels of
volatile Priority Pollutants in wastes conceivably could present a significant
health/environmental hazard which would not be detected by the EP protocol.
Interestingly, the As-contaminated groundwater sample did not have a
detectable level of volatile organics either. This result may have been influenced
by the handling of the sample prior to our receipt. The groundwater sample was
received in a partially filled plastic jug 3 months after collection. The sample
collection, storage, and shipping conditions prior to receipt are unknown.
The analyses conducted in search of PAH's and other organics revealed
that the EP extracted little or no organic material in the classes analyzed, within
the defined detection limits (1 mg/liter). In scans for PCB's, pesticides,
and other halogenated compounds there was evidence of electron-capturing material
at a concentration of approximately 0.2 ng/liter in the EP extract. The similarity
in the profiles of each corresponding sample fraction suggests background material
and not the compounds characteristic of the waste or groundwater. A further
examination of the PCB fraction of the As-contaminated groundwater by GCmass
spectroscopy revealed that any material present was below the detection limits. The
material indicated by the electron-capture detector, although unidentified at present,
is nonetheless from 100 to 1000 times lower than the maximum allowable concen-
trations suggested for pesticides.
Electron-capture detector GC profiles (Figure 5-3) of the PCB/pesticide
fractions of three samples and a PCB standard equivalent to a concentration of 0.2 ng
of PCB per liter of EP extract serve to illustrate the low concentrations of organics
present in the EP extracts. Very little material is visible in these profiles, except
for two prominent peaks in the fractions of the blank and soybean process cake.
These constituents were, however, too low in concentration for mass spectrometric
identification.
The o-nitroaniline in the As-contaminated groundwater sample was the only
organic constituent quantified from these samples. A specific measurement indicated
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37
CO
2
O
Q_
CO
Ld
cr
cr
LU
Q
fr
o
o
C
D
0
10
20 30 0 10
TIME (min)
30 40
Figure 5-3. Electron-capture GC analysis of PCB/pesticide fraction of EP
Extracts of (A) Blank 3, (B) soybean process cake, (C) scrubber sludge, and (D) a
PCB 1242 standard equivalent to 0.2 ng/liter of PCB in an EP Extract.
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38
a concentration of 320 mg/liter. No other compounds were even detected in the
measurement of o-nitroaniline, supporting the results of the analysis of the XAD-2
organic concentrates of the EP extracts.
5.5 Conclusions
Because the EP is basically an aqueous extraction of the waste, it would not
be expected to be an aggressive extractor of nonpolar organic species present in the
waste. Furthermore/ since the extraction is carried out in an open system, volatile
organic toxicants (e.g., chloroform, trichloroethylene) would be expected to
vaporize from the system .
Many organic wastes are actually liquids; therefore there may not be a need
for a more aggressive extractant, since for a fluid waste the extract is, in fact, the
waste itself. In the case of a multiphase waste, the final EP extract contains both
the original liquid portion of the waste and the extract from the solid portion. Thus
this liquid may function as a cosolvent and greatly increase the apparent aggressive-
ness of the extraction process.
If further work shows this to be a real problem, alternative equipment and
extractants might be used for the assessment of organic contaminants which may be
released in the landfill disposal of wastes. One attractive alternative may be steam
distillation.
For inorganic species we feel that the present acidic extraction is adequate.
The addition of extract blanks to the protocol is advisable to allow corrections for
background contamination due to either equipment or reagents used in the various
procedures. In some instances not running a blank may result in a false classification
of the waste as hazardous.
A comparison of the EP extracts with actual leachate samples taken from
landfills containing mixed wastes would also be desirable as a further means of
evaluation of the EP.
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39
6. AQUATIC TOXICITY
6.1 . Problem Definition
The objective of the aquatic screening tests was to determine the toxicity
and potential hazard of extracts from solid wastes to aquatic biota. The tests
used were acute (short-term lethality) tests and chronic (long-term) tests designed
to measure sublethal effects on reproduction. The test organism was the cladoceran,
D. magna, an aquatic organism sensitive to most classes of toxic aqueous chemicals.
6.2. Scope of Work
The two tests had different purposes. The acute test estimated the concen-
tration of the extract that was lethal to D. magna during a short exposure period of
48 hr, whereas the chronic test determined whether continuous exposure to extract
dilutions of 1:1 00 and 1:1000, impaired reproduction of D. magna. The chronic
test did not, therefore, establish a dose-response relationship for the extract, such
as an LCc,-,/ but only determined whether or not the extract was toxic at the con-
Ov/
centrations tested.
The procedures used are described in Appendix IV.
The parameters measured to assess the effects of chronic exposure of D. magna
to the extracts were length of life, day of first brood release, number of broods of
young produced, number of young per brood, and total number of young produced per
daphnid. Based on the results of the chronic exposure tests, one or more of these
parameters were selected as criteria for identifying those extracts potentially haz-
ardous to aquatic biota. The sensitivities of the parameters to chronic exposure to
the extracts were compared by analysis of variance combined with Duncan's Multiple
Range Test to identify significant differences between individual treatment means.
A type I error level (a) of 0.05 was set to identify significant differences between
treatment means.
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40
6.3. Results
The As-contaminated groundwater exhibited both acute and chronic
toxicity. In the two tests, the 48-hr LCcn occurred at dilutions of 2.2 and 1 .7
percent (Table 6-1). The results of the chronic toxicity tests are shown in Table
6-2. No significant effects on reproduction or survival occurred at the 1 :1000
dilution, but at the 1 :100 dilution, significant effects were found for three of the
five parameters measured. The mean number of young produced per adult and the
mean number of young per brood were both reduced by 88 percent compared with
the dilution water controls. Also, the mean day (age) of first brood release was
increased from 11 .3 for the controls to 13.7 for the 1 ;1 00 dilution.
In tests of the three wastes from a power plant, all of the extracts from the
wastes had indicated low acute toxicities (Table 6-1). The 48-hr LCrn's ranged
Ow
from 60 to 94 percent. Chronic toxicity effects on daphnid survival and reproduc-
tion were also small (Table 6-2).
The fly ash extract produced no significant adverse effects on any of the five
test parameters at either test dilution.
The scrubber sludge extract produced some significant inhibition in the
number of broods and number of young per brood when compared with the acetic acid
control; but, those effects were not significant when compared with the dilution
water controls. The inhibition in the number of young produced (compared with the
acetic acid controls) was 27 percent at the 1 :1000 dilution and 18 percent at the
1 :100 dilution, while the inhibition in number of young per brood was 32 percent and
16 percent, respectively.
The bottom ash extract was the only one of the five extracts tested to have an
apparent effect on survival of the animals in the chronic tests. At the 1 ;100 dilution,
survival (measured as an average length of life) was 14 percent less than for the acetic
acid controls. These effects were not seen at the 1 :1000 dilution. The number of
broods of young, the number of young, and the number of young per brood were also
reduced in both test dilutions, but these effects were not statistically significant.
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41
TABLE 6-1
ACUTE TOXICITIES OF As-CONTAMINATED GROUNDWATER SAMPLE
AND EP EXTRACTS TO D. magna
48-hr LC50*
c , 4 (95% fiducial limits)
Extract v '
First test
As-contaminated groundwater
Fly ash
Scrubber sludge
Bottom ash
Soybean process cake
2.2*
90 (71 to
85 (51 to
94*
*
>100)
>100)
Second test
1.7*
69*
60*
60*
§
*Concentration of the extract necessary to kill or immobilize 50% of the test
organisms in 48 hr.
*Approximate value; 95% fiducial limits could not be calculated.
^Undiluted extract had no effect on organisms.
§Less than 50% of the test organisms were killed or immobilized in 100% of the
extract.
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42
TABLE 6-2
CHRONIC TOXICITY OF As-CONTAMINATED GROUNDWATER AND EP EXTRACTS TO D. magna*
Extract and
concentration
Life length
during study
(days)
Day of
first brood
release
No. of broods
of young/adult
No. of young
produced/adult
No. of
young/brood
As-contaminated groundwoter
Control 27.2a±].8 11.3a±1.0 5.2° ±0.8 90.3° ±15.4 17.8° ±4.1
1:1000 26. la±4.2 12.5a±1.6 4.8° ±1.1 79.5a±24.3 16.5° ±3.4
1:100 28.0a±0.0 13.7b±1.0 5.0a±0.8 11.2b±I0.8 2.1b±1.7
Fly ash
Control
Acetic acid controP
1:1000
1:100
Scrubber sludge
Control
Acetic acid control^
1:1000
1:100
Bottom ash
Control
Acetic acid contror
1:1000
1:100
Soybean process cake
Control
Acetic acid controP
1:1000
1:100
25. 8C ±4.7
25.2= ±5.9
27.0C ± 3.2
27.5C ± 1.6
28. Od ± 0.0
28.0d±0.0
27.8d±0.4
28. Od ± 0.0
23. 2f ± 8.2
24. 9f ±6.9
24.5f ± 4.3
21. 4f ± 6.8
27. 4h ± 1.3
27.5h ± 1.6
27. 7h ± 1.0
27.5h± 1.6
10.8° ± 0.6
11.0° ± 0.0
11. Oc ± 0.0
11.0° ± 0.0
9.0d ± 0.0
10. 7d ± 2.3
9.0d ± 1.1
9.0d ± 0.0
8.69 ±4.7
12. Of ± 4.9
12. 8f ±0.6
12. lf ± 2. 1
11. 3h ± 1.0
11. Oh ± 0.0
11.0h±0.0
11. Oh ± 0.0
5.5C ± 1.5
5.5C ± 2.0
5.7= ± 1.1
5.7C ± 1.3
6.8d ±0.4
6.8d ± 1.0
7.2d ±0.4
6.6d ±0.9
5.0f ± 2.8
4.6f ± 2.3
4.4f ± 1.5
3.7f ±2.3
6.6h ± 1.0
6.5h ±0.7
6.8h± 1.0
6.5h ± 1.2
106. lc ± 36.2
116. Oc± 46.3
134.3C±25.3
133. Oc ± 20.2
139. 8e ±6.2
186. Od ± 39.2
134. 8e± 25.0
151. 6e± 12.6
84. 2f ± 53.0
72. Of ±40.9
62. lf ± 33.2
45. 4f ± 42.3
99.2' ± 16.3
118.5M±23.2
111.8n/'±31.4
131. 7h ± 23.7
20. Oc ±0.9
20.5= ±3.4
23. 6C ± 2.8
23.8° ±3. 2
20.7e±2.0
27.0d±3. 1
18.8e±3.6
23. 4e ±4.0
13. 5f ± 8.5
13. 5f ±7.5
13. 6f ± 5. 1
10. 2f ± 4.6
15. 1' ± 1.9
18.2h,i±2.0
16.7n/'±4. 9
20. 5h ±3.5
*Values given are mean ± S.E. Means with the same letter are not significantly different (a = 0.05).
^ Acetic acid, neutralized to pH 7.0 with NaOH, added at same concentration used in the 1:100 dilution of the extract.
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43
The soybean waste had the lowest acute toxicity of the five wastes tested.
During the first acute test, no animals were killed or immobilized after an exposure
to 100 percent of the extract for 48 hr, and in the second test less than 50 percent
were adversely affected. The low toxicity of the extract was also observed in the
chronic toxicity tests. No significant reductions in any of the five parameters
measured were observed in either of the two test dilutions.
6.4. Discussion
In the chronic tests, the effects of the waste extracts were measured on five
daphnid population parameters (Table 6-2). Of these, the most consistently
sensitive indicators of chronic toxicity were the number of young per brood and the
total number of young produced; length of life, day of first brood release, and
number of broods produced were little affected by even the most toxic sample, the
As-contaminated groundwater. Since the total number of young produced over a
given period is a function of the number of young per brood and the number of broods
produced per female, both parameters would be expected to have similar sensitivity to
these extracts.
Additional studies, still in progress, on the reproducibility of the 28-day
D. magna renewal chronic toxicity test used in this project have demonstrated the
following: (1) The mean number of young produced per brood and the mean total
number of young produced per daphnid during the 28-day test were again the param-
eters which were consistently the most sensitive to chemical toxicity. (2) When
these parameters were used as criteria for determining chronic toxicity, the 28-day
test was highly reliable and consistent in determining (a) the maximum concentration
of toxic chemical which produced no significant observed toxic effects and (b) the
minimum concentration of the toxic chemical which would produce significant
observed toxic effects. (3) When the 28-day test was repeated at intervals of
3 months for a total of three tests, the mean values obtained on each of five daphnid
population parameters, measured at five different exposure concentrations of the
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44
toxic chemical plus controls, frequently varied significantly (a. - 0.05) among the
three tests.
Therefore, the criterion selected for identification of extracts potentially
hazardous to aquatic biota was a reduction (a = 0.05) in the mean number of
young produced per daphnid during the 28-day exposure to the extracts when com-
pared with the dilution water control daphnids. Based on this criterion, only one
of the five wastes tested, the As-contaminated groundwater, would be classified as
potentially hazardous (Table 6-3). Of the remaining four wastes only one, the
scrubber sludge, showed any significant chronic toxicity; however, significant
effects were only observed when the two test dilutions were compared with the
concentrated acetic acid control. It should be pointed out that the toxicity is
probably the result of the acetate in the extraction liquids. If the toxicity is
compared with the toxicity of dilution water containing the same amount of acetate
as is present in the sample, then none of the materials tested would receive a rating
of hazardous.
From these results it is not possible to predict whether or not these wastes
would be hazardous to natural populations or communities of aquatic organisms.
There are severe limitations in the extrapolation of laboratory results based on
single-species tests for predicting effects on natural ecosystems. For example, the
test systems used in single-species tests are very simple compared with the complexity
of natural ecosystems. Thus, a significant toxic effect can be demonstrated in the
laboratory, but under natural conditions it may be mitigated, modified or even mag-
nified by the chemical, physical, and biological interactions of the ecosystem.
D. magna, however, is sensitive to most classes of toxic chemicals, and results of
toxicity tests with it will generally be conservative predictors of potential hazard to
other aquatic species.
On the other hand, there are limitations in the EP and in the D. magna
chronic toxicity test system which may result in certain types of toxic wastes not
being identified as being potentially hazardous in the protocols. All volatile
chemicals, for example, are probably lost during the EP. In addition, the
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45
TABLE 6-3
SUMMARY OF THE D. magna ACUTE AND CHRONIC TOXICITY TEST RESULTS
Extract
As-contaminated
ground water
Fly ash
Scrubber sludge
Bottom ash
Soybean process cake
48- hr LC50
(O/\
(/o)
2.2
90
85
94
No significant
acute toxic ity
Significance
of chronic
toxic ity test results
1:1000 dilution
ns
ns
ns1-
ns
ns
1:100 dilution
s
ns
ns*
ns
ns
*The criterion used to judge significant toxicity was a significant reduction (a =
0.05) in the mean number of young produced at either dilution compared with the
dilution water controls, ns, no significant toxicity; s, significant toxicity.
^Significant toxicity was not observed when the treatment means were compared
with the dilution water control mean; however, significant toxicity was observed
when the treatment means were compared with the acetic acid control mean.
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46
renewal-type test procedure used in the chronic toxicity test would be relatively
inefficient, compared with a continuous flow-through test system, for detecting
nonpersistent chemicals.
In summary, this test should only be used as a screening mechanism to
identify hazardous wastes containing toxic chemicals that can be extracted under a
specific set of conditions and that are chronically toxic to a sensitive aquatic
species under a specific set of test conditions. The test only identifies those wastes
that are potentially hazardous.
Acute toxicity tests before and after the 28-day chronic tests were done to
determine if the toxicity of the extracts had changed during the chronic tests.
Because of the low toxicities of most of the extracts, however, reliable estimates of
48-hr LCcf/s and their 95 percent fiducial limits could not be obtained. The results
Wv
(Table 6-2) suggest that all five wastes tended to increase slightly in toxicity during
the 28-day period, but the differences are probably not statistically significant.
6.5. Conclusions
The 28-day chronic toxicity test with D. magna can be used to show the
presence of toxic materials in extracts, as shown by the results with the As-contami-
nated groundwater. Daphnid reproduction, however, is greatly influenced by food
supply. The extracts often contain high concentrations of acetic acid, which is
neutralized to pH 7.0 in the tests. The acetate may be a substrate for bacteria,
which are in turn fed on by the daphnids, resulting in an increase in the number of
young produced. In three of the four extracts in which acetic acid was used, the
production of young in the acetic acid controls was substantially higher than in the
dilution water controls (without acetic acid). In addition, research indicates that
the effect of acetate on D. magna varies with concentration. Although at low con-
centrations acetate tends to increase the reproduction rate, at concentrations equal
to or greater than 0.1 percent it becomes acutely toxic. Acetic acid is even more
toxic than acetate, with an estimated 48-hr LC of about 100 mg/liter. The
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47
toxicity of acetic acid, however, is probably more a function of pH than acetate
toxicity. What is not known are the effects or interactions that acetic acid (or
acetate) may have on other toxic chemicals in the waste materials being extracted.
With respect to toxicities to aquatic biota, these effects could possibly by syner-
gistic or antagonistic, and in either case would tend to further obfuscate the
interpretation of toxicity test results. We, therefore, recommend that use of
acetic acid in the EP for aquatic toxicity be reevaluated as a screening protocol.
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49
7. MUTAGENICITY
7.1 . Problem Definition
Because the concept of toxicity includes unnatural genetic activity
(including oncogenic, mutagenic, and teratogenic activity), the Office of Solid
Waste recognizes the need for rapid and effective methods for the detection of such
activity in complex mixtures from the EP.
The bioassay protocol in this project is intended to serve as an indicator of
the chronic hazards of mutagenicity and carcinogen!city. Because of systemic
differences in the reaction to mutagenic substances, a battery of assays has been
employed by the Biology Division. The approach taken involves microbial assays
with both eukaryotic and prokaryotic organisms which detect point mutation specif-
ically and DNA damage generally.
The vast majority of known chemical mutagens are organic in nature. Thus,
because the concept of threshold is ill-defined for mutagens and carcinogens, and
because the EP extract was anticipated to be low in organic character, it was
deemed advisable to examine a concentrated extract of the organic constituents of
the EP extract as well as the extract itself.
According to the definition of a waste as a hazard (in 43 FR 58961) the
application of three assays for genetic activity would be required to delist a waste
that is listed as hazardous because of mutagenic activities. The three tests
specified are: (1) gene (point) mutations in bacteria, (2) gene mutations in eukary-
otes, either in mammalian somatic cells in culture or in fungal microorganisms,
(3) recombinogenic or repair-related phenomena. The tests selected in these
categories are (1) the Salmonella/microsome assay, (2) the Saccharomyces can/his
dual assay, and (3) the Salmonella uvrB repair assay. The Biology Division of
ORNL has had considerable experience applying the Salmonella and Saccharomyces
mutation assays to the analysis of complex mixtures, hence their inclusion.
Additionally, because of the involvement of the uvrB mutation in the design of the
Salmonella assay, the Salmonella repair assay was selected for group three.
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50
The Salmonella/microsome assay utilizes a series of histidine-requiring
mutants that revert after treatment with mutagens to the wild-type state (histidine-
independent). Generalized testing is accomplished by use of two strains, TA1537
and TA98, that detect frameshift mutagens, and two strains, TA1535 and TA100,
that detect base-pair substitution mutagens.
The Saccharomyces assay utilizes both a forward and a reverse mutation
scheme. Forward mutation is detected by the inactivation of the arginine permease
gene, leading to resistance to the toxic antimetabolite canavanine. Reverse
mutation is monitored with a histidine auxotroph which reverts by base-pair
substitution.
The Salmonella uvrB repair assay does not measure mutation per se, but DMA
damage induced by chemical treatment. The test system employs paired, identical
strains except that one (TA1978) has normal DMA repair capabilities (uvrB+) and
one (TA1538) lacks a specific step (uvrB") in the enzyme pathways responsible for
DMA repair. Preferential killing of the repair-deficient strain by the test substance
implies that the material exerts its killing effect by reacting with the cells' DMA,
and therefore may be mutagenic.
Full details of the procedures for these assays are given in Appendices V, VI,
and VII.
7.2. Results
Four EP extracts, the As-contaminated groundwater sample, and their XAD-2
concentrates have been tested in the Salmonel la/mi crosome assay, the Saccharomyces
forward and reverse mutation assay, and the Salmonella repair assay. The assays
were applied both with and without metabolic activation (both Ar- and 0B-induced
rat liver S-9 mix were used). The aqueous extracts were tested as received; the
XAD-2 concentrates were taken up in 2 ml of dimethylsulfoxide (DMSO), producing
a 250-fold (v/v) concentration of the organic material in the aqueous extracts, with
the exception of the As-contaminated groundwater, whose concentration factor was
12.5-fold.
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51
Salmonellq/Microsome Assay. The As-contaminated groundwater was
slightly mutagenic with the frameshift strain TA98, but only upon metabolic activa-
tion with Ar-induced S-9 mix (Table 7-1). The sample was not toxic at the
concentrations tested. Additionally, the XAD-2 concentrate of As waste was not
mutagenic with the missense strain TA1535 (Table 7-2). However, it was muta-
genic with the frameshift strains, TA1537and TA98, and the highly sensitive TA100
strain. It did not require metabolic activation, and the addition of S-9 mix (Ar-
or 0B-induced) reduced the mutagenic activity. It was not toxic at the dose range
tested. A dose-dependent response was elicited.
Neither the aqueous extracts nor the XAD-2 concentrates from the power
plant wastes or the soybean process cake were mutagenic with or without metabolic
activation (Tables 7-3 to 7-6). None of the materials displayed toxicity in the
dose range tested.
Saccharomyces Mutation Assay. The As-contaminated groundwater sample
was not mutagenic (Table 7-7). However, its XAD-2 concentrate was mutagenic
without metabolic activation, given a 24-hr exposure. A dose-dependent response
was observed. Metabolic activation appeared to reduce the mutagenic potential
of the XAD-2 concentrate. Neither of these test materials was toxic.
The extracts and concentrates of the power plant samples and the soybean
process cake were not mutagenic with or without metabolic activation (Tables 7-8
to 7-11). None of the material was toxic.
Salmonella DNA Repair Assay. None of the materials displayed activity in
this assay, either with or without metabolic activation (Table 7-12). The As-
contaminated groundwater was moderately toxic to both test strains (Table 7-12).
7.3. Discussion
Of the five wastes discussed in this report, only the As-contaminated
groundwater possessed detectable mutagenic activity. For the purposes of bioassay,
the mutagenic principal in the undiluted waste water is at the limit of resolution;
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52
TABLE 7-1
SALMONELLA MUTATION: As-CONTAMINATED
GROUNDWATER
Volume (jj|)
Revertants/plate
TA98
TA100
No activation
Control
0.025
0.050
0.500
5.000
59
NT*
NT
55
45
152
NT
NT
107
145
cpB activation
Control
0.025
0.050
0.500
5.000
Ar activation
Control
0.025
0.050
0.500
5.000
67
60
53
67
52
67
87
87
70
96
142
150
105
117
137
142
193
151
190
141
CNT, not tested.
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53
TABLE 7-2
SALMONELLA MUTATION: As-CONTAMINATED GROUNDWATER
XAD-2 CONCENTRATE
Volume (yl)
No activation
Control
2.5
5.0
10
25
50
75
cpB activation
Control
2.5
5.0
10
25
50
75
Ar activation
Control
2.5
5.0
10
25
50
75
Revertants/plate
TA1535
12
NT*
27
18
32
29
T*
NT
6
NT
11
15
22
13
16
TA1537
10
NT
16
41
70
94
115
NT
7
NT
15
27
44
52
69
TA98*
59
59
153
268
500
690
563
67
59
96
NT
79
206
NT
67
82
97
NT
157
292
NT
TA100*
152
151
215
414
669
963
828
142
120
150
226
288
414
590
142
155
162
212
243
295
508
Average from two or three independent experiments.
tNT, not tested.
*T, toxic.
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54
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TABLE 7-6
SALMONELLA MUTATION: SOYBEAN PROCESS CAKE EP EXTRACT'
_ . ,_. Revertants/plate
Concentration r
(fal//plate) TA1535 TAJ537 TA98 TA100
No activation
Control 15 13 24 109
50 13 13 24 87
cpB activation
Control
10
25
50
75
Ar activation
Control
10
25
50
75
6
15
13
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18
5
15
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14
19
4
16
15
15
15
17
15
15
15
16
27
37
28
27
26
30
32
30
29
19
106
85
87
91
95
93
82
97
79
83
Soybean process cake XAD-2 concentrate was highly contaminated. Filter
sterilization was not possible.
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63
TABLE 7-12
DMA REPAIR IN SALMONELLA*
% Survival
Sample and
activation
As-contaminated
ground water
None
cpB
Ar
Fly ash
None
cpB
Ar
Scrubber sludge
None
cpB
Ar
Bottom ash
None
cpB
Ar
Soybean process
cake
None
cpB
Ar
1978
EP
extract
67
70
40
120
106
88
117
104
120
51
97
108
84
109
129
uvrB+
XAD-2
concentrate
97
91
70
114
101
90
72
65
58
102
74
80
78
75
81
1538
EP
extract
42
61
78
109
93
100
68
85
92
95
81
83
126
96
110
uvrB~
XAD-2
concentrate
82
78
92
85
116
113
46
80
82
110
79
88
82
91
96
Maximum dose was 100 |-il for all samples except fly ash XAD-2, for which it
was 50 i.
-------
64
the XAD-2 concentration, however, was necessary to conclusively demonstrate
mutagenic activity. Work is currently in progress to determine if the o-nitroaniline
content of this waste accounts for its mutagenic properties.
The response of the Salmonella/microsome assay to As-contaminated ground-
water implies a frameshift mutation mechanism, which requires the addition or
deletion of DNA base-pairs. This is supported by the yeast results with this waste,
showing a moderate preponderance of induced forward mutation to can , relative to
induced reversion of the his base-pair substitution. This is typical for a response
(by this system) to a frameshifting agent.
The As-contaminated groundwater and its XAD-2 concentrate failed to elicit
a response from the bacterial DNA repair assay. There are two key considerations:
(1) the overall mutagenic potency of the waste is moderate, (2) validation studies
have shown that the repair assay is particularly insensitive (although not unreactive)
to frameshifting agents. Hence, it may not be significant to obtain a negative
result in this context.
With regard to the negative results obtained with the power plant wastes
and the soybean process cake, the aqueous extracts and XAD-2 concentrates of
these materials are extremely deficient in organic character. The majority of
organic mutagens are not detectable at the parts-per-billion level. Furthermore,
inorganic mutagens (e.g., metals and metal complexes) which might be present are
commonly not detectable by the bioassays in question.
7.4. Conclusions
Our experience has shown that biological testing within the limits of the
specific system used can be carried out with complex organic materials but
perhaps only when coupled with the appropriate analytical separation schemes. An
extrapolation to relative biohazard at this point would be, at least, premature. A
number of precautions are given below.
As noted, aqueous materials will generally contain only low amounts of
dissolved organics which may be biologically active. If the intent is to determine
-------
65
whether mutagenic components are present in a given mixture, clearly, a concen-
tration/fractionation scheme must be applied. However, the detection or perhaps
the generation of mutagenic activity may well be a function of the chemical
fractionation scheme utilized. The inability to recover specific chemical classes
or the formation of artifacts by the treatment could well corrupt the results obtained,
in addition to the possibility of an inability to detect the specific biological end
point chosen. Along with the obvious bias that could accompany the choice of
samples and their solubility or the time and method of storage, a number of biologi-
cal discrepancies can also enter into the determinations. For example, concomitant
bacterial toxicity can nullify any genetic damage assay that might be carried out.
The dose-response relationship may not be linear, and some other method for a
quantitative comparison may be mandatory. The choice of inducer for the liver
enzymes involved can be wrong for selected compounds or mixtures. Furthermore,
induction of metabolic enzymes of rat liver includes both activating and deactivating
enzymes for potential mutagens. Results with mixtures requiring activation can be
complex and different from those with pure organic compounds. Mutagenicity
studies should include not only proper metabolic activation systems but also appro*
priate quantitation of the metabolic enzymes (determined by titration studies) in the
assays. Mutagenic analyses of complex mixtures of organic constituents activated
with crude and complex enzyme homogenates require careful examination and
cautious interpretation. The choice of strain in a reversion assay could be
inappropriate for selected active components of a mixture; therefore, a battery of
tests should be considered, including an assay for forward mutation.
Additionally, the applicability of the generally used Salmonella test to
other genetic end points and the validation of the apparent correlation between
mutagenicity and carcinogen!city still remains a point to be validated through
significant fundamental research. The question of a correlation between mutagenic
potency in the Salmonella assay and carcinogenic potency should be treated with
caution. Again, the short-term assays chronically show negative results for certain
classes of organics. Similarly, compounds involved in or requiring cocarcinogenic
-------
66
phenomena would presumably go undetected. Recent studies point to synergistic
effects between compounds that may further complicate quantitative interpretation
of results with complex mixtures.
As a prescreen to aid the investigators in ordering their priorities, short-term
testing appears to be a valid approach for complex mixtures. Over-interpretation
at this stage of research, especially with respect to relative hazard or negative
results, should be avoided.
-------
67
8. PHYTOTOXICITY
8.1. Problem Definition
As part of our work, ORNL has developed and evaluated short-term phyto-
toxicity tests for use in screening potentially hazardous wastes. These tests were to
be performed on a number of wastes extracted by the EP described in Section 3.
Thus, we were in a position to identify any problems with the EP which might be
reflected in the phytotoxicity tests. Difficulties with phytotoxic effects of acetic
acid used in the EP were anticipated because of reports in the literature that acetic
9
acid, even at relatively low levels, can inhibit plant growth.
8.2. Methodology
Short-term (48 and 72 hr) root (radicle) elongation tests were performed
with radish and sorghum seeds in a controlled environment. Long-term (2 week)
seedling growth studies were carried out in pots under greenhouse conditions with
wheat and soybean seeds. Parameters measured were root length in the short-term
tests and root-shoot dry weight in the long-term tests. Treated plants were com-
pared with plants grown in distilled water in the case of the short-term tests and a
plant nutrient solution in the long-term study. The treated plants received a
10 percent concentration of the EP extract (diluted with nutrient solution) in all
the long-term tests to simulate a tenfold dilution that EPA used in its scenario to
indicate what could occur as water moves through an underground aquifer. This
groundwater could then be used to irrigate crops. In the short-term tests, treated
plants received a series of concentrations of the EP extracts. In a few cases
concentrations used in these short-term tests did not reach the 100 percent level
because of the amount of acetic acid used in the EP. Preliminary results with
radish and sorghum treated with different concentrations of acetic acid show that
plant growth is inhibited only when the acetic acid concentration exceeds
-------
68
5 ml/lifer of 0.5 N acetic acid. Further testing is being performed with wheat
and soybean.
The plant species selected for these tests are important agricultural species
and represent the two major classes (monocotyledons and dicotyledons) of flowering
plants. Because of the differences in growth habits of these two classes of plants,
they may respond differently to various chemicals in their environment. Of course,
this is also true to a lesser extent between individual species of the same class.
However, it was not feasible to test large numbers of many different species.
Detailed descriptions of the test methods are given in Appendices VIII and IX.
8.3. Results
Arsenic-contaminated groundwater was highly phytotoxic, producing a
33 percent growth reduction of radish roots (radicles) even at a 2 percent concentra-
tion. Higher concentrations (10 and 5 percent) reduced growth 70 and 59 percent,
respectively. However, at the highest dilution (0.1 percent) there was a slight
stimulation of root growth.
Data from other tests are presented in Tables 8-1 and 8-2. Table 8-3 is a
summary of the results presented in Tables 8-1 and 8-2.
None of the waste extracts tested showed toxic effects in all the tests.
Scrubber sludge was toxic to radish seeds in the root elongation test even at a
10 percent concentration, but the same concentration was not toxic to sorghum.
Fly ash, soybean process cake, and bottom ash were only slightly toxic to either
radish or sorghum at concentrations exceeding 10 percent. In the seedling growth
studies, fly ash and soybean process cake showed only a slight (but significant)
reduction of root weight but not shoot weight.
It is interesting to compare results from the short-term and long-term tests
with soybean process cake extract. In both tests the dicotyledons (radish and
soybean) were not affected. On the other hand, the roots of the monocotyledons
(wheat and sorghum) were reduced significantly. Although sorghum showed no
-------
69
TABLES-!
RADISH AND SORGHUM RADICLE LENGTH*
Extract concentration (%)
As-contaminated groundwater
Control
0.1
2
5
10
Fly ash
Control
30
Scrubber sludge
Control
10
Bottom ash
Control
100
Soybean process cake
Control
50
75
Radicle
S.D.
Radish
27± 14
31 ± 12
18± 8
11 ± 7
8± 6
31 ± 16
27± 15
20 ± 15
16± 11
22 ± 12
21 ± 11
22 ± 10
23 ± 10
length ±
(mm)
Sorghum
17± 15
15 ± 13
39 ±28
38 ±30
23 ± 13
20 ± 12
27± 12
28 ± 14
23 ± 13
% Growth
reduction
Radish
0
33*
70*
13*
12
20*
5
0
Sorghum
3
,3t
0
Length after 48- and 72-hr growth periods, respectively, at 25 C in the dark.
Controls were grown with distilled water and compared with plants treated with
the indicated concentration of extract.
'Statistically different from control at a 5% probability level.
-------
70
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71
TABLE 8-3
SUMMARY RESULTS FROM RADICLE ELONGATION BIOASSAY
AND SEEDLING GROWTH STUDIES
., . Toxic effects
_ II. Concentration
Extract and seed type , A
(/o)
Root Shoot
Fly ash
Radish*
Radish
Sorghum
Wheat*
Soybean*
Scrubber sludge
Radish
Sorghum
Wheat
Soybean
Bottom ash
Radish
Sorghum
Wheat
Soybean
Soybean process cake
Radish
Sorghum
Sorghum
Wheat
Soybean
30
10
30
10
10
10
10
10
10
100
100 §
10
10
75
75
50
10
10
yes^
no
no
yes*
no
yes*
no
no
no
no
yes*
no
no
no
yes*
no
yes*
no
no
no
no
no
-^
no
no
^^
no
no
Plants used in radicle elongation bioassay.
*5% level of significance, calculated with standard t-test by comparison of results
from treated plants with those from controls (Tables 8-1 and 8-2).
* Plants used in seedling growth studies.
^Significant growth reduction was marginal at the 100% concentration, therefore
further tests with more dilute solutions were not performed.
-------
72
effect at a 50 percent concentration after 72 hr, wheat exposed to extract for a
longer period of time was affected even though the solution was more dilute
(10 percent concentration).
8.4. Discussion and Conclusions
Because of the refinement of the root (radicle) elongation test, we are quite
confident in the test results. Radish and sorghum seeds are best suited for use in the
original design of the germination chamber, but the basic chamber can be custom
built to accommodate different seed types. We recommend that bioassays using
additional plant species be used for a more definitive screening of hazardous
materials. The main drawback in the results was caused by toxic interference of
acetic acid. Table 8-4 summarizes the effect of acetic acid on radish and sorghum
seeds in the radicle elongation bioassay. Fly ash, scrubber sludge, and soybean
process cake could not be tested at higher concentrations since extracts had to be
diluted to avoid acetic acid effects. As this problem is not inherent to the bioassay
procedure, we believe the technique is acceptable for initial screening. However,
research on the acetic acid problem is continuing with the long-term seedling
growth studies. We anticipate that some solid wastes, will require more acetic acid
in the EP than will be tolerable in these tests even after a tenfold dilution of the
extracts. In such cases it would be necessary to: (1) use more dilute concentrations
and consider tests valid only if there is growth inhibition; (2) use only data from
other tests for which the acetic acid is not a problem; (3) find suitable test plant
species which are less sensitive to acetic acid at concentrations required by the EP;
(4) use another EP; or (5) find some method to remove the acetic acid.
As is normally expected with biological systems, variability of the measured
parameters was high within and between tests. The "between-test" variation was
eliminated by running a control during each test. For radish and sorghum, control
means and standard deviations for the five tests reported were 24 ± 5 mm and
27 ± 9 mm, respectively. This high variability could be due to factors other than
just biological variability, however. It was not always possible, for example, to
-------
73
TABLE 8-4
EFFECT OF ACETIC ACID ON RADISH AND SORGHUM SEEDS IN
THE RADICLE ELONGATION BIOASSAY*
Volume of
acetic acid
(ml)
Control
1
5
10
20
Root length
Radish
40 ± 1.3
44 ± 1.3
34 ± 1.1
23 ±0.7
± S.E. (nm)
Sorghum
47 ± 1.6
48 ± 1.7
42 ± 1.2
33 ± 0.8
11 ± 0.8
% Growth reduction
Radish
0
15*
43*
Sorghum
0
lit
30*
77*
*A 0.5 N acetic acid solution was tested. pH was adjusted to 5.0 for radish
only. A second test will be performed with sorghum in which pH will be
adjusted.
^Statistically different from control at a 5% probability level.
measure plants at exactly the end of the designated growth period, though this did
not vary more than an hour or two. Temperature fluctuation during tests or between
tests could have influenced root growth. Tests are presently being performed to
quantify temperature effects on root elongation of radish and sorghum. We
recommend, however, that controls be run during each test and that care be taken
to treat controls and treatment plants exactly the same during each test to avoid
experimental errors.
Sand was chosen for the seedling growth study because various types of soil
may influence the toxicity of phytotoxic substances depending on the amount of
organic matter present. However, the attenuation by soil organic matter of
phytotoxic effects of the waste extracts or other potentially toxic substances is an
area that needs further research and must be considered in assessing the hazards of
toxic substances to the terrestrial environment.
-------
74
While these data are difficult to interpret in terms of what extracts should be
considered hazardous to the environment, they do illustrate the complexity of
developing a screening protocol for potentially phytotoxic substances. Because of
the variability among plant species in the way they respond to their environment,
large numbers of species should be tested. However, even with tests employing a
limited number of species and conditions, such as in the test presented here,
potentially hazardous chemicals may be identified or flagged for further testing.
Since all five materials tested proved to be significantly different from controls in
at least one of the tests, they are, in fact, potentially hazardous. However, final
determination of whether or not a particular material is hazardous to the environment
cannot be decided from phytotoxicity studies alone. For example, damage to plants
can arise from mutagenic changes which would not be apparent from the tests used
in the phytotoxicity studies.
-------
75
9. REFERENCES
1 . Identification and listing of hazardous waste, Section 250.13 Hazardous
waste characteristics, toxicity draft background document, December 15,
1978, U.S. Environmental Protection Agency, Office of Solid Waste,
Washington, D.C. 20460.
2. Ham, R., M. A. Anderson, R. Stegmann, and R. Stanforth. Background
study on the development of a standard leaching test, I.E.R.L., U.S.
Environmental Protection Agency, Cincinnati Ohio, 1979.
3. Junk, G. A., C. D. Chriswell, R. C. Change, L. D. Kissinger, J. J.
Richard, J. S. Fritz, andH. H. Svec. Applications of resins for
extracting organic components from water. Z. Anal. Chem. 282; 331,
1976.
4. Yamasaki, E., and B. N. Ames. Concentration of mutagens from urine by
adsorption with the non-polar resin XAD-2: Cigarette smokers have
mutagenic urine. Proc. Natl. Acad. Sci. USA, 74:3555, 1977.
5. Griest, W. H. Mu I ti component polycyclic aromatic hydrocarbon analysis
of inland water and sediment, Proceedings; International Symposium on
Analysis of Hydrocarbons and Halogenated Hydrocarbons in the Aqueous
Environment, McMaster University, Hamilton, Ontario, Canada, May 25-
27, 1978, In press.
6. Bellar, T. A., and J. J. Lichtenberg. Determining volatile organics at
microgram-per-liter levels by gas chroma tog raphy. J. Am. Water Works
Assoc., 85: 739, 1974.
7. Grob, K. Organic substances in potable water and its precursor: Part I.
Methods for their determination by gas-liquid chromatography.
J. Chromatog., 84: 255, 1973.
8. Klein, D. H., A. W. Andren, J. A. Carter, J. F. Emergy, C. Feldman,
W. Fulkerson, W. S. Lyon, J. C. Ogle, Y. Talmi, R. I. Van Hook, and
N. Bolton. Pathways of thirty-seven trace elements through [A3 coal
fired power plant. Environ. Sci. Technol., 9:973, 1975.
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76
9. Lynch, J. M. Production and phytotoxicity of acetic acid in anaerobic soils
containing plant residues. Soil Biol. Biochem., 10: 131-135, 1978
10. Upchurch, R. P., and D. D. Mason. The influence of soil organic matter on
the phytotoxicity of herbicides. Weeds, 10:9-14, 1962.
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77
APPENDIX I. Extraction Procedure
(A) Equipment
(I) An agitator which, while preventing stratification of sample and
extraction fluid, also insures that all sample surfaces are continuously
brought into contact with well-mixed extraction fluid.
(II) Equipment suitable for maintaining the pH of the extraction medium at a
selected value.
(B) Procedure
(I) Take a representative sample (minimum size 100 g) of the waste to be
tested. Separate sample into liquid and solid phases. The solid phase
is defined as that fraction which does not pass through a 0.40.5 |jm
filter medium under the influence of either pressure, vacuum, or centrif-
ugal force. Reserve the liquid fraction under refrigeration (15 C) for
for further use.
(II) The solid portion of the sample, resulting from the separation procedure
above or the waste itself (if it is already dry), shall be prepared either by
grinding to pass through a 9.5 mm (3/8 inch) standard sieve or by
subjecting it to the structural integrity procedure.
(Ill) Add the solid material from paragraph II to 16 times its weight of
deionized water. This water should include any water used during
transfer operations. Begin agitation and extract the solid for 24 ± 0.5 hr.
Adjust the solution to pH 5 and maintain that pH during the course of the
extraction using 0.5 N acetic acid. If more than 4 ml of acid for each g
of solid would be required to maintain the pH at 5, then once 4 ml per g
of solid has been added, complete the 24-hr extraction without adding
any additional acid. Maintain the sample between 2030 C during
extraction.
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78
(IV) At the end of the 24-hr extraction period, separate the sample into solid
and liquid phases as in paragraph I. Adjust the liquid phase with
deionized water so that its volume is 20 times that occupied by a quantity
of water at 4 C equal in weight to the initial sample of solid (e.g., for
an initial sample of 1 g, dilute to 20 ml). Combine this liquid with the
original liquid phase of the waste. This combined liquid, including
precipitate which later forms from it, is the toxicant Extraction Procedure
extract.
-------
79
APPENDIX II. Extractor
Apparatus
Apparatus used in the extraction is shown in Figures II-l to 11-11 .
Commercial apparatus utilized included: IEC constant-temperature centrifuge;
Millipore filtration assembly (cat. no. YY42-142-00, filter cat. no. HAWP-
142-50); Chemtrix pH controller (cat. no. 45A); Masterflex tubing pump and
pump head (cat. no. 7045-10 and 7013-00); Cole Palmer stirring motor (cat. no.
4558).
Glassware Cleaning and Reagents
Prior to extraction, all glassware and extractors were cleaned with detergent
followed by dilute nitric acid, then thoroughly rinsed with autoclaved deionized
water from a Millipore Milli-Q water purification system. The deionized water
was then autoclaved (15 psig, 127 C, 20 min) before use in cleaning and extracting.
In all cases, reagent-grade chemicals were used.
-------
80
ORNL-DWG 79-10311R
,GEARMOTOR
CONTROLLER
VESSEL
COVER-
(5) H
VESSEL
STIRRING ROD
FRONT
SIDE
Figure II-l . Extraction apparatus.
-------
81
MATERIAL' 316 STAINLESS STEEL
DIMENSIONS ARE IN INCHES
ORNL-DWG 79-I0312R
5 OD x 0.065 WALL TUBING
Figure II-2. Extracting vessel.
-------
82
ORNL-DWG 79-10313R
-------
83
ORNL-DWG 79-10314R
0.060 SHEETMETAL-
TYP
X
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9/16 DIA. BAR STOCK-
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MATERIAL-316 STAINLESS STEEL
DIMENSIONS ARE IN INCHES
Figure II-4. Upper stirring blade.
-------
84
ORNL-DWG 79-10315R
MATERIAL: PLEXIGLAS
DIMENSIONS ARE IN INCHES
5/16
BEVEL 1/16 x 45°
Figure II-5. Vessel cover.
-------
85
ORNL-DWG 79-10316R
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H DRILL THROUGH TWO PLACES
ON 53/4 B.C.
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H DRILL
NO. 7 DRILL
TAP 1/4-20 NC
SECTION A-A
2.70 REF
MATERIAL-6061 T6 ALUMINUM
DIMENSIONS ARE IN INCHES
Figure II-6. Part No. 1 .
-------
86
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-------
87
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-------
88
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-------
89
0/2.0-
Figure 11-10. Extraction apparatus.
-------
90
Figure II-l 1 . Laboratory set-up for extraction procedure.
-------
91
APPENDIX III. Analytical Methodology
EP Organic Concentrate
1 2
This procedure is based on work by Junk et al. and Yamasaki and Ames.
A 500-ml aliquot of the EP extract is adjusted to pH 6.8 with Na^PCV and to
20 mmho/cm conductance with NaCI. This preparation is then passed through 4 g
of XAD-2 (Isolab, Inc.) at a flow rate of 1 .2 ml/min. The column is rinsed with
1520 ml deionized water, and then adsorbed constituents are eluted with 10 ml
redistilled acetone. The acetone eluate is evaporated under N~ to dryness and
brought up in 10 ml cyclohexane for further fractionation. This procedure is
carried out in quadruplicate to provide duplicate samples for chemical analysis and
mutagenicity testing.
PCB's/Pesticides
The cyclohexane sample resulting from the XAD-2 procedure is fractionated
by column chroma tog raphy. It is passed through 10 g of Florisil, eluted with
150 ml of 6/1 hexane/benzene, and passed through 20 g of neutral activity III
alumina with 150 ml hexane followed by 150 ml of 6/1 hexane/benzene and 160 ml
2/1 hexane/benzene. Each fraction is concentrated to 1 ml with dry, flowing
nitrogen under reduced temperature and pressure. PCB/pesticide measurements are
made by injecting 5 uJ of the hexane concentrate into a gas chromatograph equipped
with a 10-ft OV-101 column maintained at 180C [isothermal], and detecting eluting
components with an electron capture detector calibrated with external standards.
PAH's
The three fractions obtained from the alumina step in the fractionation scheme
contain the diaromatic (hexane and 6/1 hexane/benzene) and polyaromatic (2/1
3
hexane/benzene) compounds. PAH identifications and measurements are made by
injecting 5 pi of each fraction concentrate into a gas chromatograph equipped with
a 1/8-inch O.D. x 10-ft 3 percent (w/w) Dexsil 400 on Supelcoport (100/200 mesh)
-------
92
column and temperature-programming from 100 to 320 C at 2 degrees/min. Flame
ioniration detection and calibration with external PAH standard are employed.
Other Halogenated or Polar Organics
A scan for the presence of other halogenated compounds is carried out for
each of the three fraction concentrates obtained from the alumina step in the
fractionation scheme. From each fraction, 5 u.1 is injected into a gas chromato-
graph equipped with a 10-ft OV-101 column at 180 C by use of electron-capture
detection. Further scans are set up for detection of polar compounds retained by
the Florisil and alumina columns during fractionation. These columns are further
eluted with 150 ml redistilled acetone, which is then concentrated to 1 ml with
dry, flowing nitrogen. This acetone concentrate (5 ul) is injected into a gas
chromatograph equipped with a 42-m glass capillary column (0.25 percent Carbowax
20-M) and temperature-programmed from 110 C to 200 C at 2 degrees/min. Flame
ionization detection is used.
o-Nitroaniline
A 50-ml aliquot of the arsenic-contaminated groundwater is extracted four
times with 20 ml of methylene chloride, and the combined organic layers are
concentrated to 1 ml by dry, flowing nitrogen under reduced temperature and
pressure. A 0.4-u.l sample is analyzed by GC on the Carbowax 20-M capillary
column as described above by use of an authentic external standard.
Volatile Organics
4
The procedure is a modified version of that of Grob. The aqueous sample
(5 ml) is purged with N« at 100 ml/min into a 1 cm x 1 mm precolumn packed with
2 mg Tenax (60-80 mesh) and 1 mg Florisil, in series. The volatiles are desorbed
from the precolumn in the injector (250 C) of a gas chromatograph with the analytical
column held at -70 C. The analytical column is a 50-m glass capillary coated with
-------
93
0.2 percent diethylene glycol succinate. The pollutants are detected by flame
ionization during a temperature-programmed run from 0 C to 175 C at i degrees/min
with a 32-min final hold. Quantification is carried out with external standards
treated similarly. Samples are analyzed in duplicate.
Dissolved Metals
Duplicate aliquots of the EP extracts are directly analyzed for metals by
flameless graphite furance atomic absorption spectrophotometry. Calibration is
conducted with external standards, and one sample aliquot is spiked to check
recoveries. Aliquots of EP extracts for Hg determination are preserved by addition
to a nitric acid/dichromate solution immediately after generation.
References for Appendix III
1. Junk, G. A., C. D. Chriswell, R. C. Change, L. D. Kissinger, J. J.
Richard, J. S. Fritz, and H. H. Svec. Application of resins for extracting
organic components from water. Z. Anal. Chem., 282:331, 1976.
2. Yamasaki, E., and B. N. Ames. Concentration of mutagens from urine by
adsorption with the non-polar resin XAD-2. Cigarette smokers have
mutagenic urine. Proc. Natl. Acad. Sci. USA, 74:3555, 1977.
3. Griest, W. H. Multicomponent polycyclic aromatic hydrocarbon analysis
of inland water and sediment; Proceedings; International Symposium on
Analysis of Hydrocarbons and Halogenated Hydrocarbons in the Aqueous
Environment, McMaster University, Hamilton, Ontario, Canada, May 25-
27, 1978. In press.
4. Lynch, J. M. Production and phytotoxicity of acetic acid in anaerobic
soils containing plant residues. Soil. Biol. Biochem., 10: 131-135, 1978.
-------
95
APPENDIX IV. Aquatic Toxicity Methodology
The overall protocol consisted of the following; (1) a preliminary 48-hr
acute toxicity determination; (2) a definitive 48-hr acute toxicity test; (3) a 28-day
life cycle, chronic toxicity test at two dilutions, 1 :100and 1:1000, of the EP
extract; (4) a final 48-hr LCj-n determination (this final determination was used as
a check on the EP extract to determine whether its toxicity had changed over the
duration of the 28-day life cycle test).
Acute Toxicity Tests
Laboratory cultured first instar Daphnia magna, 12 ± 12 hr old, were
utilized for acute toxicity tests. Five D. magna were exposed to 80 ml of extract
solution in 100-ml glass beakers covered with watch glasses. Temperature was
maintained at 20 ± 0.5 C in an environmental chamber under a 12-hr light/dark
regimen. The dilution water used was well water (pH 7.8, alkalinity 119 ma/liter,
hardness 150 mg/liter). The EP extracts were neutralized to pH 7.0 with NaOH.
The pH of the extract dilutions was measured at the beginning and conclusion of
each test.
Serial geometric dilutions with well water were made for each extract, with
the concentrations of each extraction solution being 60 percent of each preceding
one. All tests were run in triplicate. The range of dilutions was selected to
bracket 48-hr LC values predicted from preliminary toxicity determinations.
ou
Additional control beakers were included containing a concentration of neutralized
acetic acid equal to the highest concentration used in the acute toxicity tests.
Control beakers of dilution water without added extract were also included.
Values for 48-hr LCc^'s and 95 percent fiducial intervals were obtained by com-
puterized PROBIT analytical procedures.
-------
96
Chronic Toxicity Tests
Chronic toxicity tests were run at only two dilutions of the extract, 1:100
and 1:1000. First instar D. magna, 12 ± 12 hr old were used. One D. magna was
placed in 50 ml of extract solution in a 100-ml beaker covered with a watch glass.
Temperature, lighting, and dilution water were the same as for the acute tests.
All tests were run with ten replicates, a set of ten controls, and an additional set
of controls with neutralized acetic acid added at the same concentration as in the
1:100 dilution. The test organisms were transferred to freshly prepared test solutions
and fed 2 mg of prepared food per daphnid every Monday, Wednesday, and Friday.
The number of young and the number of broods present in each beaker were then
counted. The pH of the extract solutions was measured at the beginning and end
of each test. The tests had a duration of 28 days or until all animals had died,
whichever came first.
-------
97
APPENDIX V. Salmonella Mutagenicity Assay
The realization that the list of potential chemical carcinogens is growing
faster than our capacity to test the materials and the enormous increase in industrial
and technological activities have created an interest in short-term test procedures
for the identification of genetic hazards associated with environmental chemical
pollutants. Although the health effects of chemicals in the environment are being
extensively studied, it is obvious that short-term test procedures are necessary to
reduce the study time for evaluating the large number of potentially hazardous
substances. To control the problem of environmental carcinogenesis, greater
numbers of these compounds are to be screened and assigned priorities for further
testing. This appears to be the primary role of the short-term test. Not only
should a meaningful short-term test be faster, easier to interpret, more sensitive,
and less expensive, but it must also be reliable and relevant to the in vivo assays.
Among the various short-term assays which utilize microbial organisms, the
Salmonella test system developed by Ames has been widely used a prescreen for the
determination of genetic and potential carcinogenic hazards of complex environ-
mental effluents or products. This test system has been examined more extensively
than any other short-term assay for correlating mutagenicity and carcinogenicity.
It utilizes a series of histidine-requiring mutants that revert after treatment with
mutagens to the wild-type state (histidine independent). Generalized testing of
the compound is accomplished by use of the three strains (TAT537, TA1538 and
TA98) that detect frameshift mutagens and two strains (TA1535 and TA1 00) that
detect base-pair substitution mutagens. The design of the test is shown in Figure
V-l . A recommended protocol outlining the preparation of the components of this
2
test has been published by Ames et al. Some chemicals like dimethylnitrosamine,
certain hydrazines, and volatile liquids which are not mutagenic in the standard
plate assays are active in the modified procedure, designated the preincubation
technique. This modified procedure detects not only these compounds, but also
the majority of the compounds that have been shown to be active in the standard
plate assay.
-------
98
Aliquot of saline 0.5 ml *
or buffer »- - S-9
Molten (45'C) overlay agar
appropriately supplemented
lOul-lOOul
0.1 ml
Test, positive or solvent
control chemical
Aliquot of an overnight culture
' of bacteria (-1 V eel Is/ml)
0.5 ml
-t- S-9
S-9 mix (hepatic
homogenate from
PCB pretreated
rat plus necessary
cofactors)
,
Overlay poured on selective
bottom agar medium
Plated incubated at 37°C for 48 hours
The numbers of revertants/plate counted
1
Data analyzed
i
Interpretation/conclusion
Figure V-l . Reverse mutation assay (agar incorporation method).
-------
99
Bacterial Strains
Four Salmonella typhimurium indicator strains, TA1535, TA1537, TA98, and
TA100, are recommended for screening purposes. TA1535 and TA100 have base-pair
substitution mutation in the histidine operon; TA100 also contains an R factor which
renders the strain more sensitive to certain mutagens, possibly through error-prone
repair. TA1537 and TA98 have frameshift mutation in the histidine operon; TA98
contains an R factor and is more sensitive than TA1538. TA1537 is recommended
because of its unique sensitivity to some agents like 9-aminoacridine and certain
ICR compounds. The characteristics of these strains are shown in Table V-1 .
TABLE V-1
PROPOSED BACTERIA STRAINS*
Strain
designation
TA1535
TAT 537
TA98
TA100
Gene
affected
hisG
hisC
hisD
hisG
Additional mutations
Repair
uvrB
uvrB
uvrB
uvrB
IPS
rfa
rfa
rfa
rfa
R factor
pKMlOl
pKMlOl
"See Ames et al. for references.
Storage and Checking of Tester Strains
All strains are initially grown in nutrient broth (8 g Difco-Bacto nutrient
broth, 5 g NaCl/liter) at 37 C for 16 hr. The strains are checked for the genetic
markers in the following ways:
Histidine Requirement. Streak the cultures on minimal plates both with
and without histidine (spread 0.1 ml of sterile 0.1 M L-histidine on the agar
surface). Biotin (0.1 ml of 0.5 mM per plate) is also essential for these strains.
The strains should grow on plates containing both histidine and biotin.
-------
TOO
Deep Rough Character. A sterile filter paper disc containing crystal violet
(10 |jl of 1 mg/ml) is placed on a nutrient agar petri dish containing 0.1 ml (about
10" bacteria) of the nutrient broth culture to be tested in a thin overlay of top agar.
After 12 hr incubation at 37 C, a clear zone of inhibition around the disc (about 14-
to 18-mm diameter) indicates the presence of rfa mutation.
Presence of Plasmid. The strains with R factor (TA100 and TA98) should be
checked routinely for the presence of the ampicillin resistance. Streak a small
amount (10 u.1 of 8 mg/ml in 0.02 h>[ NaQH) of an ampicillin solution across the
surface of a nutrient agar plate. After the streak is dry, cultures to be checked are
cross-streaked against the ampicillin, and after incubation for 12-24 hr at 37 C/
strains which do not contain the R factor will show a zone of growth inhibition
around the ampicillin streak, whereas strains containing R factors will not.
Storage. Frozen permanent cultures containing fresh nutrient broth cultures
(0.8 ml) with DMSO (0.07 ml) are prepared and maintained in a Revco freezer at
-80 C. A working source of these cultures is maintained on masterplates which are
prepared as follows;
0.1 ml of sterile 0.1 iM L-histidine is spread on the surface of a minimal
glucose agar plate. After the histidine solution is absorbed by the agar, 0.1 ml of
sterile 0.5 mM biotin is added in the same way. For TA98 and TA100, 0.1 ml of
an 8 mg/ml ampicillin solution (in 0.02 N NaOH) is added. By use of a sterile
loop, nutrient broth culture of the tester strain is streaked across the agar (for TA98
and TA100, plates with ampicillin are used) and incubated at 37 C for 24 hr.
These masterplates with the cultures are stored at 4 C and can be used for several
months to grow working cultures.
Preparation of Rat Liver S-9
Male Sprague-Dawley rats (of about 180-200 g weight) are given a single
intraperitoneal injection of Aroclor-1254 at a dosage of 500 mg/kg (vehicle, corn
oil) 5 days before they are killed. They are fasted 12 hr before they are decapi-
tated and allowed to bleed. The livers are aseptically removed and washed in
cold 0.15 M KCl. All steps are performed at 0 to 4 C with cold and sterile
solutions and glassware. The livers are minced with sterile scissors in three
volumes of 0.15 M KCl (3 ml/g wet liver) and homogenized with a Potter-Elvehjem
apparatus with a Teflon pestle. The homogenate is centrifuged for 10 min at
9,000 x g, and the supernatant (S-9) is decanted and stored in convenient aliquots
at -80 C. For S-9 from 0B-induced rat livers, the same procedure as described
above is followed except that the rats are given 0.1 percent of sodium pheno-
barbital in drinking water for 1 week before they are killed.
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101
Media
Top agar (0.6 percent Difco-Bacto cigar, 0.5 percent NaCl) is autociaved
and stored in 100-ml bottles at room temperature. Before use, the agar is melted
(in an autoclave or in a steam bath), and 1 0 ml of a sterile solution of 0.5 mM
L-histidine-HCl, 0.5 mM biotin is added to the 100 ml of molten agar and mixed
thoroughly.
Complete medium (23.5 g BBL standard methods agar in 1 liter of distilled
hLO) is autociaved and dispensed into 100 x 15 mm plastic petri plates (30 ml/plate).
3
Vogel-Bonner medium E with 2 percent glucose and 1 .5 percent Bacto-
Difco agar is used as the minimal medium for mutagenesis assays and is prepared as
follows:
Vogel-Bonner Salts (SOX)
Warm distilled water 670 ml
Magnesium sulfate (MgSO/7H9O) 10 g
T" ^
Citric acid monohydrate 100 g
Potassium phosphate (K2HPO4) 500 g
Sodium ammonium phosphate 175 g
(NaHNH4PO4'4H2O)
The above salts are added to the warm water (45 C) in the specified order. Each
salt is dissolved completely before the next is added. When the salts are all
dissolved, the solution is cooled to room temperature. About 5 ml of chloroform is
added to the solution and stored in a capped bottle at room temperature.
Dissolve 15 g of Difco-Bacto agar in 1 liter of water by autoclaving. Cool
to about 60 to 70 C and add 20 ml of 50 x Vogel-Bonner salt solution and 50 ml of
sterile 40 percent glucose solution. Mix thoroughly, and dispense into 100 x
15 mm plastic petri plates (30 ml per plate). Other minimal media would presum-
ably also serve the purpose.
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102
Preparation of S-9 Mix (Activation System)
The S-9 mix contains the materials shown in Table V-2.
TABLE V-2
COMPOSITION OF S-9 MIX
1.
2.
3.
4.
5.
6.
Component
NADP
Glucose-6-
phosphate
Sodium phosphate
buffer (pH 7.4)
MgCl2
KCI
Horn oge note
Stock Volume (Ml) of
stock added/
preparation , r ,
r r ml of final mix
0.1 M
0.1 M
0.2M
0.4M
1.65M
standard KCI
9,000 x g
supernatant
40
5
500
20
20
100
Final concentration
of component
in mix (|jmoles/ml)
4
5
100
8
33
approx. 25 mg of
fresh tissue
equivalent
*Components 1 and 2 are prepared in sterile distilled water and filter sterilized
before using. Components 35 are prepared in distilled water/ sterilized, and
maintained at 4 C. Component 6 is prepared in 0.15 M KCI and stored at -80 C
until used.
Positive Control Compounds
Any assay performed should have a control in which the solvent or diluent
is employed to see its effect on the rate of spontaneous revertants. In addition to
this control, a known direct-acting mutagen and the one that requires metabolic
activation should be used to show that the assay system is working.
-------
assays.
103
The positive control compounds shown in Table V-3 could be used in these
TABLE V-3
POSITIVE CONTROL COMPOUNDS
. .. Response of strain
^»*^x««f*r\₯vnTt r\r\ *
Sodium azide
9-Aminoacridine
2-Anthramine
(uS/plate)
2.5
10.0
5.0
Activation ^^ ^ TA1537
TA100
* *
+
+ + + +
"Weak responses may be obtained,
Mutagenesis Assay by the Preincubation Method
It may be difficult to detect biological effects with the complex environ-
mental mixtures due to (1) toxicity of the complex mixture or (2) low concentrations
of the biologically active components in the complex mixture. The first problem
should be dealt with by assaying the complex mixture for general toxicity towards
bacterial survival before the mutagenesis assay is performed. The second problem
should be dealt with at the level of concentration and fractionation of the complex
mixtures. The following protocol is recommended for general toxicity.
Only one strain, TA1537,5s used to determine the general toxicity range.
Overnight culture in nutrient broth is diluted to obtain about 10 cells/ml. To
the tubes containing 2 ml standard top agar are added: 0.1 ml of the diluted
culture of TA1537, various amounts of the test material (the recommended levels
are: 1000, 500, 100, and 10 pi per tube), and 0.5 ml of phosphate buffer, pH
7.4 (for nonactivation) or 0.5 ml of S-9 mix (for activation). The contents are
mixed and poured on the surface of a bacterial complete plate. After the agar has
hardened, the plates are incubated at 37 C for 48 hr. Survival is compared with a
control plate containing solvent but no chemical. Once the toxicity is determined,
five dose levels within the 50 percent or greater survival part of the curve are
selected for actual mutagenesis assays.
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104
Preincubation Assay
Four tester strains (TA1535, TA1537, TA98, and TA1 00) described earlier are
used in the assay, and each data point is done in duplicate. The assay is conducted
as follows:
To the sterile 13 x 100 mm test tubes containing 0.5 ml of the S-9 mix placed
in an ice bath, an aliquot of the test compound (or positive control mutagen or
solvent or diluent) and 0.1 ml of an overnight bacterial culture are added. S-9 mix
should be replaced with 0,067 M phosphate buffer (pH 7.4) in nonactivation tests.
The contents are mixed and the tubes are incubated at 37 C in a shaker for 20 min.
At the end of the incubation, 2 ml of molten top agar (kept at 45 C) is added per
tube and the contents are gently mixed. The contents are then poured onto the sur-
face of a Vogel-Bonner minimal glucose agar plate (appropriately labeled). After
the agar has solidified, the plates are incubated at 37 C for 2 days and the his
revertants are recorded. Table V-4 shows the results for 2-aminoanthracene, sodium
azide, and dimethylnitrosamine tested by the standard plate incorporation method
and the preincubation method.
Revertant Confirmation
Randomly selected Salmonella revertants should be picked from plates
showing mutagenicity and confirmed for histidine independence by restreaking on
minimal plates containing no histidine.
Repeat Tests
The test on each sample should be repeated within 2 weeks following the
initial evaluation to confirm the results. The positive results obtained in the
initial evaluation with or without PCB-induced rat liver S-9, are to be confirmed
in the repeat test. If the results are negative in the initial evaluation in the
presence or absence of PCB-induced rat liver S-9, it is suggested that in the repeat
tests 0B-induced rat liver S-9 be included in addition to the PCB-induced rat liver
S-9. (It should be noted here that the liver from Ar-induced rats is the most
efficient for detecting different classes of carcinogens. The liver from 0B-induced
rats is more efficient for detection of 2-acetylaminofluorene and many other
aromatic amines, but it is very inefficient for detection of certain polycyclic hydro-
carbons.) If the repeat test results are positive in the presence of 0B-induced rat
-------
105
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106
liver S-9, they should be reconfirmed by testing the material in the presence of
0B-induced rat liver 5-9 only. If the repeat test results are negative, no further
testing is necessary. Figure V-2 gives the general scheme for evaluating the test
material in the preincubation assay for four Salmonella tester strains.
References for Appendix V
1 . McCann, J., E. Choi, E. Yamasaki, and B. N. Ames. Detection of carcin-
ogens as mutagens in the Salmonella/microsome test; Assay of 300
chemicals. Proc. Natl. Acad. Sci. USA, 72:5135-5139, 1975.
2. Ames, B. N., J. McCann, and E. Yamasaki. Methods for detecting carcin-
ogens and mutagens with the Salmonella/mammalian-microsome mutagenicity
test. Mutation Res., 31:347-364, 1975.
3. Vogel, H. J., and D. M. Bonner. Acetylornithinase of Escherichia coli:
Partial purification and some properties. J. Blol. Chem., 218: 97-106,
1956.
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107
TEST MATERIAL
Assay for General Toxicity
using TA-1537 and determine LD50
Select 5 dose levels within the 50% or
greater survival part of the curve for mutagenesis assays
Perform Preincubation assay in the presence
and absence of PCB induced rat liver S-9
Positive Results
in the presence or absence of
PCB S-9 or both
Retest using the
above scheme to confirm
the initial results
Negative Results
Retest in the presence and
absence of PCB S-9 and
Phenobarbital Induced S-9
Positive Results
with Phenobarbital
Induced S-9
Confirm the results
by retesting in the
presence of Phenobarbital
i nduced S-9
Negative Results
o May be true
negative or
0 Not enough
biologically active
component to detect
[ concentrate and/or
fractionate]
Figure V-2. General scheme.
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109
APPENDIX VI. Saccharomyces cerevisiae Gene Mutation Assay
Both forward and reverse mutation can be monitored in the haploid strain
XL7-10B. It has the genotype ap+CAN 1 hisl-7 lysl-1 ural .
Forward Mutation to Canavanine Resistance (CAN1 -»canl)
Canavanine is a toxic arginine analog to which yeast is normally sensitive.
Resistance to canavanine has been shown to be almost exclusively due to mutational
inactivation of the arginine permease. The permease gene (CAN1) has been esti-
mated to be approximately 7,700 nucleotides long, hence it offers a very large
mutational 'target.1 CAN1 is mutated by both frameshift and base-pair substitution
inducing mutagens; in addition, deletions and chromosomal rearrangements with
breakpoints in CAN1 should also be recoverable.
Reversion of hisl -7
The hisl -7 mutation is a missense mutation resulting from a base-pair substi-
tution in a histidine biosynthesis gene. This mutation confers a requirement for the
amino acid histidine. Back mutation by base-pair substitution at the original
mutant site removes the histidine requirement. Further, hisl -7 reverts by second
site mutation a second base-pair substitution at another site which 'corrects1 the
original amino acid replacement in the enzyme protein by a second compensatory
replacement. Since the reversion event is not limited to a single site, a broader
spectrum of base-pair substitutions can be detected. Also, owing to different
modes of DNA repair in yeast, hisl -7 is reverted by mutagens which have been
classified in bacterial systems as acting via a frameshift mechanism.
Both CAN1 and hisl -7 mutate readily, and the mutants are subject to a
positive selection method. Additionally, this system will tolerate a wide variety
of assay conditions (e.g., stationary phase vs. log phase cells or presence or
absence of a mammalian microsomal activation system) without requiring modifica-
tion of the mutant selection procedure or affecting the recovery of mutants.
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no
Supplies and Equipment
YPD, SC-ARG+CAN, and SC-HIS agar plates, prepoured
Steile solution of 0.067_M K2HPO4
Sterile solution of 10 percent (w/v) Na-S^CX, on ice
Clinical centrifuge and sterile centrifuge lubes
Sterile plastic test tubes with sealing caps (1 6 x 100 mm if convenient - available
from Falcon)
Shaking water bath set at 30 C (rotary preferred)
A supply of sterile 10-, 5-, and 1-ml pipettes and tips for microliter pipettor
Sufficient S-9 mix for activated assays (prepare fresh and hold on ice,
maximum 3 hr)
Ice bath for stopping assay
Sterile 0.067 M K^HPO, dilution blanks (in plastic tubes as above)
Glass bacteriaTspreader and alcohol for flaming
Alcohol or gas burner
Protective gloves for handling test materials
Test material in aqueous or DMSO solution
Hemocytometer and compound microscope
Media
Media have the compositions shown below and are sterilized by autoclaving,
YPD
1% Difco yeast extract 6 g
2% Difco-Bacto-peptone 12 g
2% dextrose 1 2 g
2% Difco-Bacto-agar 12 g
distilled water 600 ml
For broth leave out agar.
SD
0.67% Difco yeast nitrogen 4 g
base without ami no acid
2% dextrose 12 g
2% Difco-Bacto-agar 1 2 g
distilled water 600ml
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Ill
A modified synthetic complete is prepared by the following additions to SD
(concentrations in mg/liter).
sc
adenine sulfate 20
uracil 20
L-tryptophan 20
L-histidine HCI 20
L-arginine HCl 20
L-methionine 20
L-leucine 30
L-lysine HCI 30
SC-ARG+CAN is prepared by deleting arginine and adding filter-sterilized
canavanine sulfate (40 mg/liter) after autoclaving. SC-HIS is prepared by
deleting histidine.
Assay Methodology
Suspend a well-formed isolated colony of the appropriate tester strain in 0.067 M
K9HPO, and determine the cell concentration using the hemocytometer. Prepare
a dilution series and inoculate 25 ml of YPD broth with approximately 200 cells.
Grow 23 days with vigorous shaking at 30 C until late stationary phase.
Centrifuge the stationary-phase culture and resuspend in buffer. Adjust cell
concentration to 2 x 10 cells/ml.
Place sufficient tubes for the assay in the ice bath. To each tube add: up to
0.5 ml of aqueous test material (or up to 100 |jl of DMSO solution), 0.4 ml of S-9
mix (for activated assays), and sufficient 0.067 M K«HPO , to bring the volume in
each tube to 0.9 ml. Finally, add 0.1 ml of th~e~yeast suspension to each assay
tube. Seal the caps.
Without delay, place the assay tubes in the 30 C shaking water bath. At least,
a 3-hr and a 20-hr incubation should be performed.
Stop the assay by placing the tubes in the ice bath and adding 1 .0 ml of ice-cold
10 percent Na^S-CL to each tube.
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112
Plating
Plate the stopped incubation mixture directly on SC-ARG+CAN and SC-HIS (in
duplicate, 0.1 ml/plate.
Dilute the stopped mixture 10 and plate on YPD to determine survival.
Spread to dryness, flaming the spreader for each plate.
Incubate YPD plates 3 days at 30 C, others 5 days.
Count the plates. Calculate the percent survival and the mutation frequency based
on surviving titer and note the mutation yield.
Notes
. 2
Activation: 0B-induced and Ar- (or substitute) induced S-9 are used, as per Ames.
Enzyme titration; after the dose giving 50 percent survival, or the highest dose
applicable (if the substance is nontoxic), is determined, the activation system is
optimized by titration with varying amounts of S-9.
References for Appendix VI
1 . Strain available from F. W. Larimer, Biology Division, Oak Ridge National
Laboratory, Oak Ridge, Tennessee 37830.
2. Ames, B. N., J. McCann, and E. Yamasaki. Methods for detecting carcin-
ogens and mutagens with the Salmonella/mammalian-microsome mutagenicity
test. Mutat. Res., 31:347-364, 1975.
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113
APPENDIX VII. Bacterial DNA Repair Assay
DNA repair tests do not measure mutation per se, but DMA damage induced
by chemical treatment of a cell. Microbial test systems measure this damage as
cell killing. Test systems employ paired, identical cells, except one has the
normal DNA repair capabilities and one lacks a specific step (or steps) in the enzyme
pathways responsible for DNA repair. Preferential killing of the repair-deficient
strain by the test chemical implies that the chemical exerts its killing effect by
reacting with the cells' DNA, and, therefore, may be mutagenic. This implication
may not be valid in all cases, since the test cannot separate a purely lethal DNA
effect from one that also has a mutagenic component.
The following protocol describes a generalized DNA repair assay which can
utilize any of the major bacterial 'repair1 strains, i.e., the Bacillus subtilis rec -
rec pair, Escherichia coli polA -polA , or Salmonella typhimurium uvrB -
_ 0
uvrB . These systems are all based on the hypersensitivity of repair-defective
bacteria to the lethal effects of DNA-modifying chemicals.
Strain Maintenance
The source references for the strains chosen give details for the maintenance
of master cultures. The repair phenotypes are conveniently verified by checking
for UV sensitivity as follows:
The tester strains are parallel -streaked across individual nutrient agar plates
and half of each plate is irradiated with a G.E. 15 W germicidal lamp at a distance
of 33 cm. The duration of the UV exposure is 6 sec, after which the plates are
incubated overnight at 37 C. The repair-deficient strain should show growth only
on the unirradiated side of the plate, while the repair-proficient strain should show
growth on both sides of the plate.
Supplies and Equipment
Prepoured nutrient agar plates
Sterile solution of 0.067 M K2HPO4
Sterile solution of 10 percent (w/v) Na^S^CU on ice
Clinical centrifuge and sterile centrifuge lubes
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114
Sterile plastic test tubes with seaiing caps (16 x 100 mm is convenient available
from Falcon)
Shaking water bath set at 37 C (rotary preferred)
A supply of sterile 10-, 5-, and 1-ml pipettes and tips for microliter pipettor
Sufficient S-9 mix for activated assays (prepare fresh and hold on ice,
maximum 3 hr)
Ice bath for stopping assay
Sterile 0.067 M K2HPC>4 dilution blanks (in plastic tubes as above)
Glass bacteriaTspreader and alcohol for flaming
Alcohol or gas burner
Protective gloves for handling test materials
Test material in aqueous or DMSO solution
Media
Nutrient broth is composed of 8 g Difco-Bacro nutrient broth, 5 g NaCl, and
distilled water to 1 liter; sterilization is by autoclaving. Nutrient agar is nutrient
broth solidified with 2 percent Difco-Bacto agar.
Repair Assay
Prepare overnight at 37 C nutrient broth cultures of each tester strain; store at 4 C.
0.1 ml of each bacterial culture will be required for each respective assay point.
Centrifuge an adequate volume of each culture, discard the broth supernatant, and
resuspend the bacteria in a like volume of 0.067 M K0HPO,.
r 24
Place sufficient tubes for the assay in the ice bath. To each tube add: up to
0.5 ml of aqueous test material (or up to 50 pi of DMSO solution), 0.4 ml of S-9
mix (for activated assays), and sufficient 0.067 M K~HPO , to bring the volume in
each tube to 0.9 ml. Finally, add 0.1 ml of the appropriate bacterial suspension
to each of the assay tubes. Seal the caps.
Without delay, place the assay tubes in the 37 C shaking water bath. Incubate
unactivated assays for 20 min, activated assays for 2 hr.
Stop the assay by placing the tubes in the ice bath and adding 1 .0 ml of ice-cold
10 percent Na9S0O~ to each tube.
£. £* 0
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115
Plating
Prepare the following serial dilutions from each stopped assay tube: 1 :100, 1 ;10,
1:10, 1:10, using the 0.067 MK2HPO4 dilution blanks.
For each dilution, pipet 0.1 ml onto duplicate nutrient agar plates. Spread to
dryness, flaming the spreader for each plate.
Incubate the plates inverted at 37 C overnight.
Count the plates. Calculate percent survival for each strain at each assay point,
relative to untreated controls.
References for Appendix VIII
1 . Kada, T., K. Tutikawa, and Y. Sadaie. In vitro and host-mediated "rec-
assay" procedures for screening chemical mutagens; and phloxine, a
mutagenic red dye detected. Mutat. Res., 16: 165-174, 1972.
2. Slater, E. E., M. D. Anderson, and H. S. Rosenkranz. Rapid detection of
mutagens and carcinogens. Cancer Res., 31: 970-973, 1971.
3. Ames, B. N., J. McCann, and E. Yamasaki. Methods for detecting carcin-
ogens and mutagens with the Salmonella/mammalian-microsome mutagenicity
test. Mutat. Res., 31:347-364, 1975.
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117
APPENDIX VIII. Seed Germination/Radicle Length Assay
Two levels of tests were completed on EP extracts from the following solid
wastes: fly ash, scrubber sludge, soybean process cake, and bottom ash. Level one
studies consisted of a root elongation bioassay using radish (Raphanus sativus L.
'Early Scarlet Globe1) and sorghum (Sorghum vulgare var. saccharatum ' Sugar Drip1)
seeds. In previous tests seeds were germinated in petri dishes and root (radicle)
lengths of treatment and control results were compared. However, the time required
to measure root lengths was so great due to their coiled growth pattern that it was
not practical to use enough seeds for good statistical comparisons. Therefore, we
developed special vertical germination chambers which took advantage of the
geotropic growth response of plants resulting in straight hypocotyl growth and a
tenfold reduction in measurement time.
One approach to reduce variability was to sieve seeds to separate them into
size categories. U.S.A. standard testing sieves numbers 8, 10, and 12 with
openings (in millimeters) of 2.36, 2.00, and 1.70, respectively, were used for
separation. Within a test only one seed size was used for controls and test dilutions.
Although 200 seeds were used for each treatment, only 150 seeds were actually
measured. The excess allowed for nongerminating seeds and for radicles which
were less than 5 mm long.
The germination chambers were constructed of 3-mm-thick Plexiglas
(Fig. VIII-1) with inside dimensions of 10 cm high x 1 .5 cm wide x 71 cm long.
The size of the chambers was determined by the size of the incubator in which they
were to be used. Chambers were mounted on a Plexiglas base support. Two
pieces of 3-mm-thick Plexiglas were cut to an appropriate size to fit inside a
chamber but extended above the chamber sides about 3 cm for convenience in
handling. One hundred depressions (drilled with an electric drill and bit) spaced
at 2-cm intervals in staggered rows 2 cm apart across one of the Plexiglas sheets
served as seed counters, seed spacers, and to help hold the seeds in place. Seeds
were placed on the Plexiglas sheet and brushed into the depressions. A piece of
blotter paper was saturated with the solution to be tested and pressed firmly against
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118
ORNL-DWG 78-13072
COVER
INDENTATION
FOR SEEDS
BLOTTER PAPER
LEACHATE
Figure VIII-1. Germination chamber.
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119
the seeds until impressions were seen. Additional test solution, up to a total of
100 ml for 48-hr tests and 125 ml for 72-hr tests, was added to the germination
chambers. We recommend that the blotter paper be saturated with the test solution
in a flat tray rather than by standing on edge in the chambers, since standing on
edge could result in differential movement of chemicals up the paper causing chroma-
tographic separation and variable doses to seeds at different positions. The second
Plexiglas sheet was positioned so that the seeds and blotter paper were sandwiched in
between the two sheets of Plexiglas, which were then taped securely on the sides
and top and placed vertically into the chamber. A Plexiglas lid was placed on top
to reduce evaporation. The entire apparatus was then placed in an unlighted
incubator set at 25 C. Since the tests reported here were run a small fan has been
installed in the growth chamber to exhaust volatiles. The fan affected the chamber
temperature, thus thermostat adjustments have been necessary. During this period
of adjustment, tests have continued, to avoid delay. Since controls are run with
each test, we were not concerned about effects of these temperature differences
between tests. However, tests are presently being conducted to quantify tempera-
ture effects on root elongation of radish and sorghum. We used two chambers
(200 seeds) for each test solution or for each concentration of a particular solution
and two chambers containing distilled water as controls. After a predetermined
time period (48 hr for radish, 72 hr for sorghum) the chambers were removed from
the incubator and the root lengths were measured with calipers.
For cleaning, the chambers were filled with an appropriate cleaning solution
(0.1 N HCI) and allowed to stand until their next use, when they were rinsed with
distilled water. The rest of the apparatus was washed with two pipette washers, one
containing 1 N HCI and one connected to a distilled water supply for rinsing.
Acetic acid was used in the EP to maintain the pH of the extract at approxi-
mately pH 5.0. Since acetic acid is toxic to plants, the highest concentration of
extract used in the root elongation test was the concentration having less than
5.86 ml/liter of 0.5 N acetic acid. In preliminary tests this and higher volumes
of the organic acid were toxic to radish seeds (Table 8-4).
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120
The material referred to as the As-contaminated groundwater sample was
not carried through the EP, but was diluted directly from the original solution for
the root elongation tests. Since this particular waste was extremely toxic (based
on an initial test with radish seeds) and safety problems were not yet resolved,
further study was not undertaken.
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121
APPENDIX IX. Seedling Growth Assay
The long-term seedling growth studies were conducted with wheat (Triticum
aestiyrum 'Bear') and soybean (Glycine max 'Bedford') by use of the following
protocol. Seeds were soaked for approximately 1 ir in deionized water. To 1 liter
of sand (white silica sand which passes through a 2;> mesh sieve was added 350 ml of
a 10 percent concentration of the extract plus one tablespoon Purina Plant Food per
gallon of solution; this was to ensure ample exposi re of plants to extract. There
were 50 wheat seeds in each of 5 containers and 1q
containers, giving a total of 250 and 150 seeds, respectively.
Plants were exposed to a 10 percent concerj
soybean seeds in each of 10
tration of extract in droplet form.
The dose was sufficient to restore loss by evapotranspi ration. The amount of time
between each application ranged from every other pay to every 3 days. Constant
pressure was applied via a compressed air tank to mastic bottles containing the test
solution. Solution was forced through Tygon tubirg to a polyethylene nozzle
(inverted buchner funnel). The volume was regukted with a screw clamp adjusted
to a flow rate of 6 ml/sec. Solenoid valves conne cted to a pushbutton timer
delivered the solution for 10 sec when engaged.
disposed of or acid-washed in order to assure inexpensive, readily available
component parts which are easily cleaned between test runs.
Wheat plants were grown for 2 weeks and soybean for 3 weeks. At harvest,
sand was washed from the roots, roots and shoots w ;re separated, and dry weights
were recorded. To reduce variability between sanples 5 soybean plants and 10
wheat plants were consolidated. The N value eqialed as many as the number of
sample groups available. A standard t-test was used for comparison of treated and
control weights.
his design is simple and can be
it US GOVERNMENT PRINTING OFFICE 1979-640-079/196
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