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Prepublication issue for EPA libraries (Publication No. 844)
and State Solid Waste Management Agencies
TOXICITY OF LEACHATES
This report (SW-844) describes work performed
for the Municipal Environmental Research Laboratory,
Office of Research and Development under interagency agreements
and is reproduced as received from the contractor.
The findings should attributed to the Oak Ridge National Laboratory
and not to the Municipal Environmental Research Laboratory.
Copies will be available from the
National Technical Information Service
U.S. Department of Commerce
Springfield, VA 22161
U.S. ENVIRONMENTAL PROTECTION AGENCY
1980
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2d printing by the Office of Solid Waste,
with minor changes to the title page, page iv, and the
foreword from the report (EPA 600/2-80-057) published by
EPA's Office of Research and Development in March 1980
This report was prepared by Oak Ridge National Laboratory under
interagency agreements.
Publication does not signify that the contents necessarily reflect
the views and policies of the U.S. Environmental Protection Agency,
nor does mention of commercial products constitute endorsement by
the U.S. Government.
An environmental protection publication (Shelf no. 844) in the
solid waste management series.
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ABSTRACT
This report represents a multidisciplinary effort to establish a data base for
evaluation of the toxicity of extracts from solid wastes representative of various industries.
Seventeen solid wastes and an arsenic-contaminated groundwater were studied.
The solid waste samples were subjected to the extraction procedure proposed in the
Federal Register on December 18, 1978 (43 FR 58956), and the resulting extracts were
characterized.
Analytical chemical methodologies were evaluated to determine their suitability
for use in the analysis of the waste extracts, and procedures were established for preparing
organic concentrates from the extracts. None of the extracts contained appreciable
organic material. The groundwater sample was heavily contaminated with o-nitroaniline.
Six of the wastes had metal concentrations in excess of the standard proposed in Section
3001, Subtitle C of the Resource Conservation and Recovery Act (PL 94-580).
Screening assays for toxicity to aquatic organisms and terrestrial plants were
evaluated for use in chracterization of waste extracts. Four extracts exhibited toxicity
to aquatic organisms at the proposed limit dilution. Fourteen wastes showed phytotoxic
effects distinguishable from control values.
Short-term in vitro mutagenicity bioassays were evaluated and applied to testing
the waste extracts. Only the arsenic-contaminated groundwater possessed mutagenic
activity.
This report was submitted in fulfillment of DOE-IAG-40-646-77,
EPA-IAG-78-DX-0372 by the Oak Ridge National Laboratory under the sponsorship of
the U. S. Environmental Protection Agency. This report covers the period April 1, 1978,
to May 18, 1979, when work was completed.
111
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CONTENTS
Abstract ill
List of Abbreviations v
Acknowledgments v
1. Introduction 1
2. Summary and Conclusions 2
3. Recommendations 5
4. Samples and Extraction 6
5. Chemistry 23
6. Aquatic Toxicity 44
7. Phytotoxicity 58
8. Mutagenicity 79
References . . .- 92
Appendices 94
A. Extraction Procedure 94
B. Extractor 96
C. Experimental Protocols for Chemistry 108
D. Materials and Methods for the Aquatic Toxicity Screening Tests 112
E. Radicle Length Assay 114
F. Seedling Growth Assay 117
G. Salmonella Mutagenicity Assay 118
H. Saccharomyces Cerevisiae Gene Mutation Assay 127
I. Bacterial DNA Repair Assay 131
iv
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LIST OF ABBREVIATIONS
AAS atomic absorption spectrometry
Ar A roc I or
BAP benzo(a)pyrene
EP Extraction Procedure
EPA U. S. Environmental Protection Agency
GC gas chromatography
NOEC "no observed effects" concentration
ORNL Oak Ridge National Laboratory
PAH polyaromatic hydrocarbon
PCB poly chlorinated biphenyl
cpB phenobarbital
RCRA Resource Conservation and Recovery Act
ACKNOWLEDGMENTS
The investigators wish to acknowledge the technical assistance provided by the
following individuals during the course of this study: From Analytical Chemistry Division,
R. R. Reagan, S. H. Harmon, and R. W. Harvey; from Environmental Sciences Division,
G. P. Wright and J. L. Forte; from Biology Division, A. A, Hardigree, B. E. Allen,
D. W. Ramey, and L. R. Dry.
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FOREWORD
The Environmental Protection Agency was created because of Increasing public
and government concern about the dangers of pollution to the health and welfare of the
American people. Noxious air, foul water, and spoiled land are tragic testimonies to
the deterioration of our natural environment. The complexity of that environment and
the interplay among its components require a concentrated and integrated attack on the
problem.
Research and development, that necessary first step in problem solving, involves
defining the problem, measuring its impact, and searching for solutions. The Municipal
Environment Research Laboratory develops new and improved technology and systems to
prevent, treat, and manage wastewater and solid and hazardous waste pollutant discharges
from municipal and community sources; to preserve and treat public drinking water
supplies; and to minimize the adverse economic, social, health, and aesthetic effects of
pollution. This publication is one of the products of that research; it is a most vital
communications link between the researcher and the community.
This report presents results from a laboratory investigation conducted to establish
a data base for evaluating the toxiciry of extracts from solid wastes representative of a
variety of industries. Analytical chemical methods, screening assays for aquatic toxicity
and phytotoxicity, and short-term in vitro mutagenicity bioassays were evaluated and
applied to waste extracts.
Francis T. Mayo, Director
Municipal Environmental Research Laboratory
V1
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SECTION 1
INTRODUCTION
Under Subtitle C of the Resource Conservation and Recovery Act (RCRA) of 1976
(PL 94-580), the Environmental Protection Agency (EPA) is required to promulgate
regulations for the management of hazardous waste. To assist the EPA in developing
characteristics for identifying wastes posing a potential hazard to human health and the
environment, the Oak Ridge National Laboratory (ORNL) has conducted studies on
leaching of toxicants from various solid waste materials, analytical procedures for
characterizing extracts of wastes, and screening bioassays for evaluating the toxicity of
extracts. This final report summarizes work during the period April J, 1978, through
May 18, 1979. Experimental work during this reporting period in support of the Office
of Solid Waste's development of the proposed Extraction Procedure (EP) (43 FR 58956) has
concentrated on:
(1) extracting 17 wastes of diverse origin by use of the EP to delineate 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 the aquatic organism Daphnia
mag no and terrestrial plants;
(6) determining the utility of these procedures by use of the extracts prepared
from the 17 wastes and from a sample of groundwater contaminated with arsenic.
Prior to the reporting period of this report, scoping studies were conducted using
distilled water extracts (prepared at the University of Wisconsin) from 10 wastes. In
addition, extracts were prepared at ORNL from municipal refuse, sewage sludge, and an
arsenic-contaminated sludge. Four extractants were used: distilled water, acetate
buffer, HCI acidified water, and Netherlands mix. The results of initial chemical
characterization and environmental and biological toxicity testing of these materials
were presented in a draft report to the Office of Solid Waste on May 2, 1978. It was
in light of this early experience that the program described in this final report evolved.
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SECTION 2
SUMMARY AND CONCLUSIONS
A number of experiments were conducted using a selection of industrial wastes in
order to assist the EPA in developing and evaluating the EP proposed on December 18,
1978 (43 FR 58956). The studies consisted of four parts:
(1) Conducting the EP on 17 industrial wastes to uncover any problems inherent in
the proposed EP and to gain experience in its application on a variety of waste types.
(2) Evaluating available analytical methods and analyzing the EP extracts for a
variety of contaminants including heavy metals and organic compounds.
(3) Conducting range-finding studies under the direction of EPA's Office of Solid
Waste to operationally define the EP. These involved experiments on agitation
techniques, methods of adjusting pH, and preliminary studies on toxicity of extractants.
(4) Developing and evaluating screening tests for use- in evaluating EP extracts for
additional properties of mutagenicity, aquatic toxicity, and phytotoxicity.
The chemical analysis work was designed to evaluate protocols for identifying and
quantifying selected heavy metals and organic compounds identified by the EPA as
indicators of hazard. For heavy metals, the methods employed were those described in
Methods for the Analysis of Water and Wastes, EPA-600/4-79-020, U.S. Environmental
Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, OH
45268. For organic compounds, a variety of methods were employed including XAD resin
concentration and the Bellar-Lichtenstein sparging technique. A preliminary validation
of the methodology for the inorganic species was obtained through an inter laboratory
reproducibility study.
No significant problems were encountered in extracting or analyzing wastes for
inorganic species. Due to either the ionic character of the extraction fluid (water
acidified with acetic acid), the low concentration of organic species in the wastes
examined, the open extractor used, or possible adsorption on the filter membranes, no
appreciable organic material was found in the waste extracts.
In order to develop a short-term screening test suitable for use in determining the
potential toxicity of waste extracts to aquatic organisms, acute and chronic toxicities to
the cladoceran D. magna were studied. Acute tests determined the concentration of the
EP extract that was lethal to the organisms during a 48-h exposure. Of the wastes
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tested, only the extract from the soybean process cake and gasification waste No. 1
exhibited no significant acute toxicity.
Chronic assays were conducted at extract dilutions of 1:100 and 1:1000, and
effect on organism fecundity was used as the measure of toxicity. Five criteria were
utilized to determine reproduction effects in the chronic tests. The mean number of
young and the number of young per brood produced by each adult were the two criteria
found most consistently sensitive for identification of toxicity. Toxicity assessment of
the EP extracts used the number of young produced. Toxic effects were noted for four of
the wastes tested at either one or both concentrations.
Short-term phytotoxicity tests were studied for use in determining if use of
irrigation water contaminated by leachate from the waste posed a potential hazard to
food crops. Two types of tests were studied, root elongation using radish and sorghum
seeds, and seedling growth using wheat and soybean seeds. For the root elongation
(radicle length) test, a chamber was developed in order to permit the precise
measurement of radicle length in a timely manner. The longer-term (2-week) seedling
growth studies used root and shoot dry weight and plant length as the measures of growth.
Studies were performed to determine the optimal growth medium, methods of test solution
application, and toxic effects of the waste extracts. Of the 18 samples tested, 14
proved to be significantly different from the controls in at least one of the tests.
However, any conclusion relative to potential toxicity must be tempered by the
observation that the acetate used in the EP exhibited significant phytotoxicity. Also,
in a field situation soil attenuation could affect the apparent toxicity of leachates.
These leachates were tested in the absence of soil.
The proposed Section 3001 regulations make use of a variety of short-term
mutagenicity assays in the delisting procedures. In order to determine their utility,
three assays diagnostic for genetic activity were evaluated by use of the available waste
extracts and the arsenic-contaminated groundwater. The assays studied and the types of
genetic activity they are known to detect are;
(1) Salmonella/microsome-- point mutation in bacteria
(2) Saccharomyces canr/his+ — gene mutations in a lower eukaryote
(3) Salmonella uyrB —repair of DMA damage.
Of the 18 samples examined, only the arsenic-contaminated groundwater
possessed detectable mutagenic activity. This material elicited a positive response in
both the Salmonella/microsome and Saccharomyces assays. The bacterial DMA repair
assay did not respond to the arsenic-contaminated groundwater sample. With regard to
the negative results obtained with the remaining wastes, the extracts and even the XAD
concentrates were extremely deficient in organic character. Furthermore, inorganic
mutagens, which might have been present, are not detectable by the assays studied.
This study has identified a number of areas in which further work is required.
These are:
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(1) Reproducibility of the EP. Preliminary studies indicate it has a coefficient of
variation in the range of 2 to 94% for elements such as Ca, Cr, and Ni.
(2) Effect of sample storage on EP extract. A potential problem was noted with
loss of sample moisture affecting EP extract results.
(3) Extractor-induced contamination. Experiments conducted to evaluate this
potential problem found that the level of contamination is related to the pH of the
extraction and the abrasive ness of the waste. However, the maximum contamination
noted was at least an order of magnitude below the level at which a waste would be
determined to be hazardous under the proposed regulations (43 FR 58956).
(4) Possible retention of organic compounds on the filter media during filtering of
the waste or the extracted liquid prior to evaluation of the extract.
(5) Effect of various types of agitation on integrity of solid wastes undergoing
extraction.
(6) Methods of determining the phytotoxicity and aquatic toxicity of substances
teachable from solid wastes in the presence of acetate or methods of measuring
teachability which would not pose the problem encountered with the presence of acetate.
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SECTION 3
RECOMMENDATIONS
Based on our observations, ORNL makes the following recommendations.
(1) Sample and extract storage specifications should be added to the regulations,
such as those recommended in Methods for the Analysis of Water and Wastes,
U. S. Environmental Protection Agency, Environmental Monitoring and Support
Laboratory, Cincinnati, OH 45268, and in the draft report Handbook for Sampling
Hazardous Waste, California Department of Health, Berkeley, CA.
(2) A nonmetallic extractor, such as one fabricated from either
polytetrafluoroethylene or glass could be used to lessen the possibility of contamination
occurring during waste extraction.
(3) Alternative extraction procedures should be evaluated to increase the
aggressiveness of the EP toward organic substances. One possible candidate may be the
steam distillation procedure examined during the course of this work.
(4) Authentic landfill leachates should be compared to laboratory-generated
extracts in order to assess the environmental relevance of the EP.
(5) Because of the confounding effects that acetate has on phytotoxicity studies,
a procedure to determine phytotoxicity should be developed that does not employ acetic
acid. Additional plant species should be considered for incorporation in the
phytotoxicity testing battery.
(6) Because the presence of acetate can stimulate reproduction of the D. mag no
and acetate could have synergistic or antagonistic effects, additional studies should oe
performed to determine the effects acetate has on aquatic organisms; if necessary, a
procedure should be developed to correct these problems.
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SECTION 4
SAMPLES AND EXTRACTION
Seventeen solid wastes were extracted by use of the proposed EP published in
43 FR 58956 (see Appendix A). These samples represent various industrial wastes and
coal combustion and gasification residues. Table 1 contains a listing of the waste samples
and extraction dates. The arsenic-contaminated groundwater sample was distributed for
testing as-received and was not extracted by the EP. Detailed information regarding the
chemical and physical characteristics of the wastes was not provided with the samples.
TABLE 1. EXTRACTION DATE AND EXTRACTOR MATERIAL FOR WASTE SAMPLES
Extraction Extractor
Waste date material*
Arsenic-contaminated groundwater — —
Soybean process cake 10/18/78 1
Metal processing waste 10/27/78 1
Plater's waste 11/02/78 1
Raw shale 11/28/78 2
Retorted shale 11/30/78 2
Dye waste 12/05/78 2
Textile waste 12/08/78 2
Municipal sewage sludge 03/06/79 3
Power plant No. 1 fly ash 09/22/78 1
Power plant No. 1 bottom ash 10/20/78 1
Power plant No. 1 scrubber sludge 10/03/78 1
Power plant No. 1 treated scrubber sludge 03/31/79 3
Power plant No. 2 fly ash 03/13/79 3
Fluidized bed residue 02/07/79 3
Gasification waste No. 1 10/06/78 1
Gasification waste No. 2 10/19/78 1
Gasification waste No. 3 11/02/78 1
*1, Plexiglas vessel and stirrer; 2, Type 316 stainless steel vessel and stirrer; 3, Type
316 stainless steel vessel with Teflon stirrer.
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REPRESENTATIVE SAMPLING
All waste samples received for testing were stored outdoors in an open shed.
This was a safety precaution deemed necessary due to lack of information concerning the
nature of the samples.
We do not assume the waste samples to be necessarily representative of their
respective solid wastes or industrial sources. The samples were supplied by EPA through
other sources, and those sources refused all requests for additional information on the
samples. Because of an extended storage time, and no information on collection and
storage factors such as collection procedures for the samples, date of collection,
temperature changes, and possible sample-container interactions, the samples must be
evaluated as-received solely on the basis of their individual characteristics as
determined during the course of analysis and evaluation at this laboratory.
It is unreasonable to assume or predict changes (biological, chemical, and
physical) that may have occurred in a sample over time. These potential changes,
though, should be a consideration in the final version of the hazardous waste
characterization procedure.
No universal procedure was 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 and
quartering was utilized.
PRELIMINARY SAMPLE DATA AND COMMENTS
Preliminary laboratory data collected on the waste samples (Table 2) included:
(1) Solids content, given as a fraction of the weight of the sample after
drying per as-received sample weight.
(2) General physical description, including color, odor, and
consistency.
As indicated by the data, solids content was highly variable among samples,
ranging from 1.0000 to 0.2436.
A problem was identified in the extraction protocol with regard to the moisture
content of individual wastes. After initial separation of a waste into liquid and solid
phases by the methods of filtration or centrifugation, a considerable fraction of the solid
weight was, in some cases, bound liquid. For example, the apparent weight of a 100-g
solid sample containing 25% moisture by weight is not comparable to 100 g of dry
sample. Leachate contaminant concentrations may be affected by differing moisture
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TABLE 2. SAMPLE CHARACTERISTICS AND TREATMENT
Waste
Solids
*
content
Physical state
Sample treatment
before extraction
Soybean process cake 0. 4090
Metal processing waste 0.4105
Plater's waste 0.2436
Raw shale 0.9991
Retorted shale 0.9997
Dye waste 0.4249
Textile waste 0.7053
Municipal sewage sludge 0.7359
Power plant No. 1 0.9923
fly ash
Power plant No. 1 0. 7442
bottom ash
Power plant No. 1 0.6844
scrubber sludge
Power plant No. 1 treated 0.7803
scrubber sludge
Power plant No. 2 fly ash 0. 9983
Fluidized bed residue 0. 9999
Gasification waste No. 1 0. 9938
Gasification waste No. 2 1.0000
Gasification waste No. 3 0.9999
dark grey; moist, so! I-1 ike
consistency and odor
blue-green; moist, clay-like
consistency and odor
orange sludge topped with large
amount of orange liquid
grey; hard granular particles,
heterogeneous mixture of large
and small particles with the
majority being <3/8-in., odorless
black; fine powder; odorless
purple; moist, clay-like consistency
light grey gelatinous solid with
small amount of standing white
liquid; fibers and small fabric pieces
were mixed with waste; obnoxious
odor
dark brown; moist, soil-1 ike
consistency
dark grey; powder-like; dead insects
and dried plant materials mixed with
waste; odorless
dark grey; heterogeneous mixture of
large and small particles; odorless
dense, light grey sludge topped with
small amount of standing liquid;
cement-like odor
dark grey sludge; clay-like
consistency; odorless
grey; powder-like; odorless
light and dark grey; gravel-like
particles; odorless
charcoal grey; fine with some
larger agglomerates present
black; glassy, irregularly shaped
particles with sharp edges
charcoal grey and brown;
irregularly shaped particles;
large agglomerates present
none
none
supernatant removed by centri-
fugation prior to extraction,
solid:liquid ratio = 0.606:1
(by wt.)
sized to pass 3/8-in. sieve
none
none
supernatant removed prior to
extraction, solid:liquid ratio=
7:1 (bywt.)
none
none
sized to pass 3/8-in. sieve
supernatant removed,
sol id: liquid ratio = 6.5:1
(bywt.)
none
none
none
none
none
sample sized to pass 3/8-in.
sieve
Weight of sample after drying per original weight of sample.
8
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contents and will not necessarily remain constant under varying conditions of collection
time, transportation, and storage, due to evaporation.
EP AND COMMENTS
The extractions were executed by use of various extractor designs and materials.
Designs and materials were progressively changed as problems arose.
Initially, the extractions were performed with four Plexiglas extractors borrowed
from the Environmental Engineering Department of the University of Tennessee. Due to
the possible problems of abrasion and solubility in organic media associated with the use
of Plexiglas, -EPA recommended the use of Type 316 stainless steel materials.
Extractors constructed of this material were then supplied to ORNL by EPA.
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
with hard, granular wastes such as bottom ash. The design was similar to the Plexiglas
extractor and is given in 43 FR 58956. The suitability of the extractor was pretested
with a hard, granular solid-. This type of waste had presented stirring problems in the
Plexiglas extractor. Similar problems occurred with this prototype. The major problems
included: binding of the stirring blade and solid particles, movement of the vessel,
stalling of the stirring motor, uneven blade alignment, and sample grinding. This unit
was not used for any of the reported sample extractions.
As a result of these problems, a modified version of the extraction equipment was
designed and fabricated at ORNL, incorporating EPA's recommendations. As requested
by EPA, all parts exposed to waste or extract were fabricated of Type 316 stainless steel.
Details of materials and design are given in Appendix B, Figures B-l to B-l 1. As shown
in the figures, the most important improvement in the modified design is the unitizing of
vessel, supporting frame, and stirring motor. The integration of the vessel, support
stand, stirring rod, and stirring motor is viewed as the best way of assuring positive
alignment of the agitator blade and vessel bottom. This feature minimizes the
probability of waste particles binding 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 because 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.
The new extractor design eliminated many of the previous problems. However,
the main problem remaining was that of sample grinding (particle size reduction) when
the particle size was approximately 1/4 in. Extractor designs having a fixed blade near
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the vessel bottom would be expected to permit sample grinding. The spacing would
determine which particle size is susceptible to this action. To eliminate sample grinding,
the secured stainless steel blade was replaced with a Teflon stirrer shaft with two moveable
blades that swing out when rotated (Cole-Palmer cat. no. 6367-42). The Teflon stirrer
blade permitted similar stirring action, eliminated sample grinding, and avoided binding.
Table 1 contains a listing of the extractor design used for each waste sample.
An inherent problem associated with the use of a Type 316 stainless steel extractor
is the dissolution of metals from the vessel wall and stirring blade. Due to the low
concentrations of contaminant being analyzed, a noncontaminating extractor material
should be utilized. An extractor material such as Type 316 stainless steel has the
potential of introducing metals such as Cr, Ni, and Zn into the extract. The extent of
this contamination is discussed below under Extraction Procedure Blanks and in Section 5.
To avoid the problem of extract contamination by extractor material, it is recommended
that Teflon or glass materials be used.
Due to the lack of specification in the EPA proposed EP, the laboratory practices
and utilization of reagents cited in Appendix B were deemed adequate. We recommend
the specification of water and chemical grades as an addition to the EP.
EXTRACTION DATA AND COMMENTS
Data recorded during the extraction of the solid wastes are represented in Table 3.
The following parameters were recorded for each individual extraction apparatus:
(1) initial pH,
(2) final pH,
(3) amount of 0.5 N acetic acid initially added to adjust the solution
pH to 5,
(4) total 0.5 N acetic acid added during the 24-h extraction period,
(5) electrical conductivity of the final extract.
The solution pH was automatically adjusted during all extractions.
EXTRACT DATA AND COMMENTS
Following extraction, the solid and liquid phases were separated by use of a
Millipore filtration system. A Millipore (Type HA) 0.45-nm pore size filter was utilized
for final filtration. In all cases, vacuum filtration proved to be satisfactory for all
wastes extracted to date. Research activities concerning the filtration of EP extracts can
be found at the end of this section.
Data recorded upon completion of the extractions are given in Table 4. The
following parameters were recorded for the extract in its final form:
10
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•^J-Tf-^-Tj- OOOCO
s *. ^ «.
— — CN —
-2 8
Q. Z M- U_ u
o "^J" ""^ ^o o *o • — ^ r —
^in-C'^i'in CN^^CO
CN CN CN CN CN
00000 OOOO
ooooo oooo
ooooo oooo
ooooo oooo
ooooo oooo
ooooo oooo
O--CNOO OOOOIN.OO
^^" t-O ^O '-O ^O ^^ ^f ^^ ^4"
o — CN o o iN.inmo
•^•inininin ^ ^f ^ •^-
c ' c
1 — CNCO^n) — CN CO
-------
TABLE 4. EP EXTRACT DATA
Waste
Soybean process cake
Metal processing waste
Plater's waste
pH
5.20
5. 11
6.95
Electrical
conductivity
(y mhos/cm)
240
3, 100
7,000
Total acetic
acid added
(meq/g sample)
0.077
1.41
1.22*
Color
transparent,
yellow
transparent,
colorless
translucent,
orange
precipitate
addition of
prec ipitate
Remarks
immediately formed after
the supernatant; the
was filtered out, and the
eluate, precipitate, and supernatant
were all sent to Analytical Chemistry
Raw shale
Retorted shale
Dye waste
Textile waste
Municipal sewage sludge
Power plant No. I
fly ash
Power plant No. 1
bottom osh
Power p ant No, I
scrubber sludge
Power plant No. 1 treated
scrubber sludge
Power plant No. 2 fly ash
Fluidized bed residue
Gasification waste No. 1
Gasification waste No. 2
Gasification waste No. 3
5. 15
7.53
5.03
7.02
5.07
4.80
5.00
4.87
5.0
4.9
12. 11
5.05
4.88
5. 19
3,300
5,750
1,600
5,200
1,620
1,910
154
2,340
2,600
»
480
10,000
190
33
193
1.37
2.0*
0.53
1.75*
0.24
0. 191
0.030
0.429
0.715
0.04
2.0*
0.00
0.00
0.054
transparent,
colorless
transparent,
colorless
opaque, dark
blue
translucent,
white
transparent,
yellow
transparent,
colorless
transparent,
colorless
transparent,
colorless
transparent,
colorless
transparent,
colorless
transparent,
colorless
transparent,
colorless
transparent,
colorless
transparent,
colorless
Division
white precipitate formed after addition
of supernatant to leachate; the
precipitate
precipitate
separately
Division
was filtered out, and the
and the filtrate were sent
to Analytical Chemistry
'Maximum amount allowed (4 ml acid/g solid).
14
-------
(1) pH,
(2) electrical conductivity,
(3) total 0.5 N acetic acid added,
(4) color,
(5) remarks on unusual appearance or occurrence.
After the EP was completed, aliquots of the extract were delivered to appropriate groups
at ORNL for analysis. When delivery could not be accomplished immediately after
completion of the procedure, all extract samples were refrigerated at 4°C.
EXTRACTION REPRODUCIBILITY
Sample extract variability was investigated between the extracting vessels utilized.
Four 2.5-1 vessels were utilized for each sample extraction to produce at least 8 I of
combined sample EP extract. An aliquot of EP extract was reserved from each vessel
before the extracts were combined and distributed for testing. These aliquots were
analyzed separately for Cr, Ni, and Ca concentrations, to provide information concerning
the variability associated with each extraction. Concentrations of Cr and Ni were
investigated because of the possible contamination with these metals from the extracting
vessel. Concentrations of Ca were determined because of the excellent sensitivity and
reproducibility associated with analytical Ca concentration determinations.
The coefficient of variation was calculated for each sample investigated as a
function of the Cr, Ni, and Ca concentrations analyzed (Table 5). (Coefficient of
variation values normalize sample variation by expressing the standard deviation as a
percentage of the mean, for comparison purposes.) As these values indicate, the
variability ranged from 2 to 94%; however, no overall trends were evident.
Figures ] to 3 graphically describe the high and low concentrations, mean, and
reported values for the EP concentrations of Cr, Ni, and Ca. These figures generally
indicate that the EP variability trend exceeds that expected for the analytical
determinations. An investigation of the percent variabilities associated with analytical
analysis is recommended.
The high degree of variability found for each sample extraction indicates the
need for further research in the area of EP reproducibility. Possible explanations for
sample-to-sample variation have been discussed in previous sections, such as the lack of
information concerning sample collection and storage, and the solids content of the waste
samples. The temperature, length of storage, type of container, and pH all have the
potential of affecting the variability of extract contaminant concentrations. Further
research is needed to investigate the sources and degree of variability associated with
EP variables and extract analysis.
-------
TABLE 5. SAMPLE EP EXTRACT CONCENTRATIONS OF CHROMIUM, NICKEL,
AND CALCIUM BETWEEN VESSELS UTILIZED, EXPRESSED IN TERMS OF
THE COEFFCIENT OF VARIATION*
Coefficient of variation (%) for
Extract Cr Ni Ca
Soybean process cake
Metal processing waste
Plater's waste
Raw shale
Retorted shale
Dye waste
Municipal sewage sludge
Power plant No. 1 bottom ash
Power plant No. I scrubber sludge
Power plant No. 2 fly ash
Gasification waste No. 1
Gasification waste No. 2
Gasification waste No. 3
43.61
16.61
53.06
63.70
6.75
72.20
2.36
61.39
12.80
82.59
14.30
60.70
61.65
87.01
13.90
28.03
38.27
19.84
53.66
9.57
94.35
19.10
11.18
9.31
47.30
41.51
11.11
7.84
13.95
9.43
6.51
28.92
2.56
5.33
4.43
4.23
4.80
27.86
14.97
Coefficient of variation = (S.D./mean) X 100.
EP BLANKS
As previously discussed Type 316 stainless steel for the extractor materials has the
potential of contaminating EP extracts. Water blanks alone only provide indications of
possible background contamination. Solution pH and sample consistency also have been
found to significantly affect contamination of EP extract blanks.
To determine if solution pH affects the leaching of metals from the extractor,
water blanks were run at two additional pH values. These blanks were adjusted to
pH 3.5 with 0.1 N nitric acid and to pH 10 with 0.1 ammonium hydroxide. Table 6
contains the results of the inorganic analysis of the blanks for these pH values. The
acidic blank contained significantly higher concentrations of Cd, Cr, Cu, Pb, Ni, and
Zn than did the alkaline blank.
An additional blank study investigated the effect of sample consistency on extract
contamination over the 24-h extraction time. Two types of blanks were run without pH
adjustment; one with water and the other with acid-washed sand at a 1:20 solid:solution
ratio. Water samples were collected at each point where water was handled before it
16
-------
ORNL-DWG 79- (4336
2
(O3
z
o
S 10'
(0"
1—' LOW VALUE
i—i HIGH VALUE
— MEAN
• AGO VALUE
^W'
EP EXTRACTS
Figure 1. Range and mean chromium concentrations
in four separate EP extracts and the Analytical
Chemistry Division value after the extracts were
combined, for each sample extraction.
17
-------
(0«
g
<
z
o
10'
ORNL-OWG 79-14337
I I I
'
'
•—' LOW VALUE
r-. HIGH VALUE
— MEAN
• ACD VALUE
*>>' * **
EP EXTRACTS
Figure 2. Range and mean nickel concentrations in four
separate.EP extracts and the Analytical Chemistry
Division value after the extracts were combined, for
each sample extraction.
18
-------
F1 I T
E
2
UJ
10°
ORNL- DWG 79- <4338
LOW VALUE
HIGH VALUE ~
MEAN
COMBINED SAMPLE VALUE
I-
iju
/V *** 4? ^ XVVV ^ ^/V5
4' ^ ^' ^ <^<^
EP EXTRACTS
Figure 3. Range and mean calcium concentrations
In four separate EP extracts and the concentration
value after the extracts were combined, for each
sample extraction.
19
-------
TABLE 6. INORGANIC ANALYSIS OF EP
BLANKS AT PH 3.5 AND 10
Concentration (ng/ml)
Element
Ag
As
Ba
Be
Cd
Cr
Cu
Hg
Ni
Pb
Sb
Se
Tl
Zn
Blank at
pH3.5
<0.03
0.23
<0.2*
<0.01
0.30
37.0
52.0
0.68
107.0
0.76
<0.5
<5.0
<1.0
45.0
Blank at
pH 10
<0.03
0.09
<0.2*
<0.01
0.019
6.1
4.0
0.42
6.4
<0.3
<0.5
<5.0
<1.0
0.57
Concentration in u.g/ml.
reached the Type 316 stainless steel extractor. Additional samples were collected after
initial extractor contact, and after 1, 3, 7, and 24 h of stirring. The unfiltered samples
from each point were analyzed for Cr and Ni concentrations. Figure 4 contains a plot of
the results.
The results of the blank study illustrated in Figure 4 clearly indicate an increased
contamination of extracts when a hard granular solid, such as sand, is extracted. Also,
over the 24-h extraction time, increasing amounts of extractor contaminants were found.
The filtration step in the EP tends to remove a major fraction of the contaminants.
Further work is needed to determine if generalizations can be made concerning the
removal of abraded metal particles by filtration.
The results of the additional blank studies clearly indicate problems associated
with contamination of EP extracts by Type 316 stainless steel extractor materials.
Solution pH and sample consistency provide variation relating to this problem. The
background level of contamination from the extractor is not constant and does not seem to
20
-------
500 -.
200 -
100 -
50 -
J 20 -
10 -
5 -
8
ORNL-OWG 79-14339
1
2
3
4
5
6
7
8
9
—
~~
™~
~"~
-
-
-
—
-
-
_
"•
-
FROM MILLIPORE MILLI-0 WATER
PURIFICATION SEPTEM
FROM WATER COLLECTING FLASK
AFTER AUTOCLAVING WATER
FROM EXTRACTOR- INITIAL
FROM EXTRACTOR - 1 HOUR
FROM EXTRACTOR -3 HOURS
FROM EXTRACTOR-7 HOURS
FROM EXTRACTOR -24 HOURS
AFTER FILTERING
1 1 1 1 1 1 1 4 1
— HgO BLANK »Cr VALUES '»
- -SAND BLANK A Ni VALUES j *
/ \
/ \
/ \
/ \
1 ; x
/ \
/ 1
\i \
i * i
'i !
// '
Ml
•
/'/ \
$ ^\
A/\
il
I / i i i i i i i
1 23456789
—
-
—
~
—
—
-
—
-
-
-
-
—
2 -
1 -
0.5 -
0.2 -
0.1
1 2
WATER COLLECTION POINTS
Figure 4. Water and sand blank chromium and
nickel concentrations at successive water
collection points. Type 316 stainless steel
extractors were used with Teflon stirrers.
21
-------
be predictable. Therefore, we recommend the use of noncontami noting materials such
as glass or polytetrafluoroethylene in order to minimize this effect.
FILTRATION OF EP EXTRACTS
Other research activities within ORNL's Environmental Sciences Division have
been examining the effect of filtering aqueous solutions containing polyaromatic
hydrocarbon (PAH) compounds. Filters studied include those specified in the EP (i.e.,
Millipore, type HAWP, 0.45-u.m pore size, and Nuclepore 0.4-u.m pore size membrane
filter). Preliminary data indicate a substantial loss of PAH compounds upon filtration.
For example, as much as 99% of certain PAH compounds is absorbed by Millipore filters.
Due to the preliminary status of the research, the complete data will not be presented
here. When the research is complete, data will be presented in the open literature.
This type of information, though, may help explain why some expected organic compounds
have not been found in Millipore-filtered EP extracts reported in this document.
22
-------
SECTION 5
CHEMISTRY
INTRODUCTION
The role of the Analytical Chemistry Division in this project was twofold. The
primary role was to develop, validate, and apply cost-effective analytical methods for the
identification and quantification of selected chemical constituents in the groundwater
sample and the 17 EP extracts. Potentially toxic/mutagenic constituents for identification
and measurement (Table 7) were selected from the U.S. EPA Primary Drinking Water
Standards and the Priority Pollutant List; they included certain trace elements, volatile
organics, halogenated organics, and PAHs. The organic constituents were concentrated
from the aqueous samples by a macroreticular resin sorption technique. They were
fractionated by adsorption column chroma tog raphy and identified and measured by gas
chromatography (GC). Inorganic constituents were determined directly by atomic
absorption spectrometry (AAS).
The secondary role of the Analytical Chemistry Division was to support mutagenesis
biotesting in the Biology Division by preparing organic concentrates of the groundwater and
EP extracts to concentrate potentially mutagenic constituents into a form more compatible
with bioassay. The same macroreticular resin concentration procedure as employed in the
analytical studies was used for the bioassay preparations.
In addition to the development and application of analytical methodology, a small
scoping effort was also conducted on alternate solid waste extraction procedures suitable
for extracting organic materials from the waste.
DEVELOPMENT AND VALIDATION OF ANALYTICAL METHODOLOGY
Early analytical studies of aqueous extracts of solid wastes indicated that the
organic constituents of interest were present at very low concentrations and that they could
be quite complex in nature. Direct analysis was not a viable approach. Thus, a means
of efficiently concentrating the organics from the aqueous matrix and of separating them
into chemical classes was needed for the successful identification and quantification of the
selected organic constituents. A study was also conducted to compare the commonly used
AAS with other techniques.
23
-------
TABLE 7. SPECIFIC COMPOUNDS SELECTED FOR MEASUREMENT IN EP
EXTRACTS AND GROUNDWATER SAMPLE
PCB/
PAH Volatile organic Element pesticide
Acenaphthene
Fluoranthene
Napthalene
Benz(a)anthracene
Benzo(a)pyrene
Chrysene
Anthracene
Benzo(ghi )pery I ene
Fluorene
Phenanthrene
Dibenz(a,c and a,h)anthracenes
Pyrene
1,1-Dichloroethylene Ag
Methylene chloride As
trans-l,2-Dichloroethylene Ba
Acrolein Be
Dichlorobromomethane Cd
Tetrachloroethylene Cr
Bromoform Cu
Bis[2-chloroethyl] ether Hg
Acrylonitrile Ni
1,1 -Di chloroethane Pb
Trichloroethylene Sb
2-Chloroethyl vinyl ether Se
1,1,2-Tri chloroethane Tl
Chlorobromomethane Zn
S-Tetra chloroethane F
Ar-1242
Lindaney-
isomer
Methoxychlor
Endrin
Toxaphene
\
Plus 31 other PAHs not included on the Priority Pollutant List.
Preparation of Organic Concentrates
2,3
Solvent extraction and macroreticular resin concentration with XAD-2 resin
were compared for their efficiencies in recovering organics from an aqueous matrix. A
mock EP extract was made from 500 ml of triply distilled water acidified with 1 .7 ml of
glacial acetic acid and spiked with 3 x 105 to 6 x 105 disintegrations/min activity of a
'^C-labeled tracer. The pH was then adjusted to 6.8 with NasPO^ and the conductance
was adjusted to 20 mmho/cm with NaCl. The mock EP extract was extracted 5 times with
100-ml portions of organic solvent or was passed through a disposable cartridge containing
4 g of XAD-2 resin (Isolab, Inc., Akron, Ohio) (after reference 2), and the sorbed
organics were eluted by the method detailed in Appendix C. Recoveries of the tracers
were determined by standard liquid scintillation spectrometric methods. Table 8 shows
the compounds tested and the recoveries. Although solvent extraction, particularly with
methylene chloride, appears to be the more effective extraction method, it should be noted
that the use of methylene chloride in bioassay preparations is questionable. The XAD-2
resin procedure provides a direct 100-fold concentration, and thus the results are not
strictly comparable with those for solvent extraction. The results for solvent extraction
would have to be adjusted downwards to include the effects of a 100-fold concentration
step; therefore there may be little actual difference between the solvent extraction and
XAD-2 resin recoveries. Because of the comparable efficiencies of the methods and the
24
-------
TABLE 8. COMPARISON OF SOLVENT EXTRACTIONS AND XAD-2 RESIN SORPTION
FOR RECOVERY OF ORGANICS FROM EP EXTRACTS
Compound
Hexadecane
Naphthalene
Benzo(a)pyrene
Ar-1254
Stearic acid
Succinic acid
Phenol
Sitosterol
Stearyl alcohol
Indole
Solvent
Cyclohexane
100
100
100
100
100
0
3
82
4
41
Percent recovery
*
extraction
Methylene chloride
100
100
100
100
99
0
99
100
95
95
XAD-2
resi n *
93
100
82
95
91
0
70
91
90
84
w
Extraction recovery only.
Adsorption/desorption and direct 100-fold concentration recovery.
fact that the XAD-2 resin procedure can be applied to as many as 15 samples simultaneously
by use of a simple peristaltic pump, we chose the XAD-2 resin concentration procedure as
the protocol method for recovery of organics from the EP extracts for both chemical
analysis and mutagenic bioassay. Four 500-ml aliquots of each EP extract were subjected
to the XAD-2 resin concentration procedure as soon as possible after receipt of the EP
extract from the Environmental Sciences Division. EP extracts and concentrates were
stored at 4°C in the dark. Two of the four concentrates were chemically analyzed, and
two were delivered to the Biology Division for mutagenesis bioassay.
Fractionation and Analysis of Organic Concentrates
The organic concentrates from the EP extracts must be separated into simpler, more
well-defined fractions prior to analysis. To accomplish this task in a cost-effective
manner, we employed a modified version of our existing adsorption column chromatography
procedure4 for isolation of PAHs from environmental materials. Figure 5 shows the
fractionation procedure and the recoveries of radiotracers at various steps. This work was
carried out on cyclohexane extracts of mock EP extracts early in the project, but the
recoveries beyond the initial solvent extraction should be very similar for organic
concentrates prepared with XAD-2 resin. The specific details of the procedure are
25
-------
ORNL-BIO. 36541
500 ml acetic acid blank
(pH 6.8, conductance ~20 millimhos)
extract 5 X 100 cyclohexane
I
150ml 6:1 hex/benz
ll%Indole
8% Stearyl alcohol
78% Hexadecane
80% Napthalene
86% BAP
100% PCBS
I
cyclohexone extract
94% Stearyl alcohol
41%Indole
100% Hexadecane
82% Sitosterol
100% BAP
100% PCBS
100% Napthalene
10 g Florisil
extracted water
100% Phenol
100% Succinic acid
'31% Indole
6% Stearyl alcohol
4% Sitosterol
150 ml acetone wash of Florisil
64% Stearyl alcohol
20% Indole
6% Hexadecane
50% Sitosterol
14% BAP
150 ml hexone
46% Hexadecane
30% Napthalene
96% PCBS
2% Stearyl alcohol
20 g alumina
*
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. Recoveries of tracers in the fractionation procedure.
26
-------
included in Appendix C. A preliminary separation of the organic concentrates into
nominally nonpolar and polar fractions was accomplished on a Florisil column. The
nonpolar fraction was further subdivided into a poly chlorinated biphenyl (PCBJ/pesticide/
monoaromatic/diaromatic/paraffin fraction, a diaromatic fraction (containing the bulk of
the diaromatics), a polyaromatic fraction, and a heteroaromatic fraction on an alumina
column. Each of these five fractions was concentrated to 1.0 ml and analyzed by GC
under the conditions described in Appendix C for the specific constituents chosen and also
for any others migrating at the same time. The polar fraction was also screened on the
packed Dexsil 400 column for higher-boiling constituents not observed on the Carbowax
capillary column, and the diaromatic and polyaromatic fractions were examined with the
electron capture detector GC for halogenated and other electronegative constituents.
4
The parent procedure from which this procedure is derived has been validated for
PAH analysis of freshwater sediments and is being evaluated5 for stream and river water.
Two EPA Laboratory Performance Evaluation Standards for PCBs are being analyzed by this
procedure.
Analysis of o-Nitroaniline
Tracer studies indicated that the XAD-2 resin procedure is not particularly effective
for collection of ionized organics. Thus, the o-nitroaniline content in the arsenic-
contaminated groundwater sample was extracted with methylene chloride under conditions
more rigorous than those specified in the EPA Level 1 Environmental Assessment Manual0
and was measured by GC as described in Appendix C. Four extractions were sufficient to
remove all solvent-extractable color from the groundwater sample.
Analysis of Volatile Organics
Volatile organic compounds in the EP extracts are present at too dilute
concentrations for direct aqueous injection GC. However, they are readily identified
and determined by the well-established purge and trap procedure. We adopted the
manual version described in Appendix C. The apparatus is shown in Figure 6.
The procedure was evaluated with aqueous standards prepared immediately before
analysis with the following toxicants: 1,1-dichloroethane, 1,2-dichloroethane,
hexachloroethane, trichloroethane, tetrachloroethane, 2-chloroethyl vinyl ether,
chloroform, 1,1-dichloroethylene, 1,2-dichloropropane, 1,3-dichloropropylene,
bis[2-chloroisopropyl] ether, bromoform, dichlorobromomethane, trichloroethylene, and
dichloromethane. Because of the potentially high losses of these standards from an
aqueous sample, the standard was made up at 1 mg/ml in ethylene glycol, and aqueous
dilutions were prepared only immediately prior to purging. Thermal desorption and GC
analysis of the trapped standards indicated an average precision of ±20%. While the
absolute recovery of each toxicant is unknown, recoveries should be highly reproducible
for standards and samples treated in the same manner. Sensitivity is approximately
0.1 mg/l when a 1.0-ml sample is analyzed, and greater sensitivity can be achieved by
27
-------
Teflon Stopper,
ORNL DWG 79-13149
-Tenax Capsule
- Condenser
0-Ring with
w~i\ i i iy »» i 1.11
Nylon Screen y
/
He(N2)
IN
• Aqueous Material
Figure 6. Purge and trap apparatus.
28
-------
purging a larger aliquot of sample. This procedure also is being tested with EPA
Laboratory Performance Evaluation Standards. Aliquots of the EP extracts are taken
immediately into bubble-free vials after the last step in the EP procedure and are stored
in the dark at -20°C until analysis.
Analysis of Metals
There are several instrumental methods available for determining the content of
selected metals (Table 7) in the EP extracts. Because AAS is the most commonly
available method, we conducted a comparative study of flameless graphite furnace AAS
with five other methods, including spark source mass spectroscopy (with isotope dilution
where possible), inductively coupled plasma emission spectrometry, optical emission
spectrometry, and instrumental neutron activation analysis. An EP extract of a sewage
sludge rather than a standard made up in deionized water was chosen as the test material
for comparison of the methods. The sludge should more realistically model some of the
interferences to be encountered in the EP extracts of a wide range of solid wastes. The
results of quadruplicate analyses by each method (listed in Table 9) indicate that AAS
compares well with the other methods, and suggest that it is a valid method for analysis
of EP extracts.
Supporting evidence for the validity of our AAS method was obtained in a
collaborative study with Mr. Frank Schmidt of the Illinois State EPA. Results for
quadruplicate analyses of an EP extract of sewage sludge by AAS in both laboratories are
compared in Table 10. Considering the concentration levels, the results are consistent
and in generally good agreement. The results for the four footnoted metals did not agree
in the first round of the study, but were in agreement in the second round (shown in Table
10). It is likely that inadvertent contamination of the EP extract during packaging for
the first shipment caused the disparity in the first round.
Final validation of our AAS method was obtained by analysis of two EPA
Laboratory Performance Evaluation Standards for As, Be, Cd, Cr, Cu, Hg, Ni, Pb, and
Se. The results were biased slightly low but were acceptably accurate and precise.
Fluoride also was determined in the EP extracts by a specific ion electrode
procedure. Because of the lesser importance of fluoride, the procedure did not receive
additional validation beyond that in set-up and routine quality control.
Samples of the EP extract for metals analysis were collected in acid-washed glass
containers immediately after the last step in the EP. Separate aliquots for Hg
determination were preserved with nitric acid and potassium dichromate.
29
-------
Element
TABLE 9. COMPARISON OF ANALYTICAL METHODS FOR
METALS IN EP EXTRACT OF SEWAGE SLUDGE
Average concentration ± S.D. (u.g/1) for method*
AAS
ID-SSMS
SSMS
ICPS
OES
NAAf
Ag
As
Be
Cd
Cr
Cu
Ha
Ni
Pb
Sb
Se
Tl
Zn
0.15±0.01
30 ±0
0.41 ±0.02
1,200 ± 0
25.0±0.8
700 ± 10
0.026 ±0.001*
3,400 ± 190
31±3
99.0±1.5
<2
12.0±1.4
36»700 ± 678
4.
1,100± 0
<200
750 ±70*
4,1 00 ± 270
<45
39, 000 ± 2,200
<20
<20
10
£10
£10
<20
800 ±80
<;500
700 ±70
3,000 ± 300
<100
45,000 ± 4,000
<10
78 ±3
<100
1,030± 30
58±6
41±5
<20
55, 000 ± 1,500
*AAS, atomic absorption spectrometry; ID-SSMS, isotope dilution-spark source mass
spectroscopy; ICPS, inductively coupled plasma emission spectrometry; OES, optical
emission spectrometry; NAA, neutron activation analysis. Results are for quadruplicate
measurements unless otherwise indicated.
tn=3,
*n=2.
The results from
ANALYSIS OF EP EXTRACTS
A total of 18 wastes and 12 blanks were subjected to the EPA EP.
the chemical analysis of these EP extracts are presented below.
Organic Compound Analysis
A summary of the organic constituent analyses and the estimated limits of detection
of each analysis is presented in Table 11. For the volatile organics, a few mi Hi liters of
EP extract were collected immediately after the last step in the EP and stored in a freezer
until analysis. In spite of these precautions in the collection and preservation of
volatile constituents, no volatile organics were detected above a 0.1 mg/l level. This
undoubtedly resulted from volatilization losses during the EP 24-h stirring of the wastes in
unsealed containers. Use of a sealed container purged with an inert gas through a solid
30
-------
TABLE 10. COMPARISON OF ORNL AND ILLINOIS STATE
EPA ANALYSES OF EP EXTRACT OF SEWAGE SLUDGE*
Average concentration ± S.D.
in study by
Element ORNL Illinois State EPA
AgJ
As1"
Bat
Be
Cd
Cr
Cu
HO*
Ni
Pb
Zn
0.29
6. 25 ±0.22
<500
0.41 ± 0.02
1,200* 0
25 i 0.8
700 ± 10
0.027*
3,400 ± 190
31 ±3
36,700 ± 678
<5
7
400
<5
1,100±0
38 ± 5
830 ± 50
<0.03
4,000 ± 0
41 .5 ± 1
48,500 ± 1,730
Quadruplicate determinations except where indicated.
Redetermined in second round of comparative study.
adsorbent trap should allow collection and analysis of the volatile organics released from a
solid waste during the EP.
Analysis of the arsenic-contaminated groundwater sample did not reveal any
detectable volatile organics by the time the analysis was conducted. This result probably
was influenced by the handling of the sample prior to our receipt. It arrived in a
partially filled plastic jug 3 months after collection, and the conditions of collection,
storage, and shipping prior to receipt are unknown. Ample opportunity existed for loss
of volatiles.
The analyses for the other protocol organic constituents revealed very little
extracted organic material within the limits of sensitivity and the compound classes
examined. Nothing was detected which would even approach a "toxic" RCRA
classification based on ten times the concentrations listed in the EPA Drinking Water
Standards (Table 12). We feel that this is a result of the ionic nature of the EP extractant
fluid, in which primarily hydrophilic species would be extracted, leaving the bulk of the
essentially hydrophobia species unextracted. The latter would include most of the
organic compounds on the Interim Primary Drinking Water Standards and the Priority
31
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32
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TABLE 12. SELECTED ORGANIC AND INORGANIC CONSTITUENTS IN THE EPA
INTERIM PRIMARY AND PROPOSED SECONDARY DRINKING WATER STANDARDS*
Standard
Interim primary
Secondary
*
Source; Federal
None set.
Constituent
Ag
As
Ba
Cd
Cr
F
Hg
Pb
Se
Endrin
Lindane
Methoxychlor
Toxaphene
Cu
Zn
Register, 43(232), 59019 (Dec. 18,
Maximum concentration (pg/l)
50
50
1,000
10
50
t
2
50
10
2
4
100
5
t
t
1978).
Pollutants List. In addition, there is some evidence (e.g., reference 9) that membrane
filtration, such as that used in the RCRA protocol EP, can effectively adsorb organics from
aqueous solution.
However, there was some indication of organics in the EP extracts. All samples
contained PCBs at levels below 1 ng/l, most of which probably originated from the system
blank and not the waste sample itself. Figure 7 shows electron capture detection GC of
an Aroclor (Ar)-1242 standard and the PCB/pesticide fraction from the EP extract of the
fluidized bed residue. The GC profiles correspond nearly identically, and the PCB
concentration in the EP extract is estimated to be approximately 0.2 ng/l.
Identification was based on the GC profile; PCB concentrations were too dilute for
successful mass spectroscopic identification.
Some nonprotocol constituents were identified and measured in the samples. A
few EP extracts were found to contain n-paraffins, as reported in Table 13; these were
limited mainly to n-C2()H42 through n-C-25^52' altnou9n traces of both heavier and
lighter homologs were present. The paraffins represent the most concentrated organic
compounds identified in the EP extracts, but their concentrations do not appear to reflect
the expected organic compound content of the wastes. The observation that the EP
33
-------
ORNL- DWG 79- 13017
B
TlME (MINI)
TEMPfC)
Figure 7. Gas chroma tog ram of (A) Ar-1242 standard equivalent to 0.2 ng/1 and
(B) PCB/pesticide fraction from EP extract of fluidized bed residue.
34
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extract of fly ash contained higher concentrations of paraffins than those of textile waste
or sewage sludge, and the similarities in the distributions of the various paraffins, suggest
a common source. However, none of the EP extract blanks contained paraffins.
A specific determination was made of the o-nitroaniline content of the arsenic-
contaminated groundwater. Because we were unsure of its recovery from water in the
XAD-2 resin procedure, we used a more efficient methylene chloride EP. GC analysis
indicated an o-nitroaniline concentration of 320 mg/l. No other organic compounds
were detected in the analysis, supporting the results obtained with the XAD-2 organic
concentrate of the groundwater sample. This result for o-nitroaniline groundwater
concentration far exceeds the maximum permissible EP extract concentration level
(30 mg/l) calculated from rat oral LD5Q data obtained from the NIOSH Register/,^ and
it is the only organic compound data which could result in a "toxic" RCRA classification,
based on the proposed delisting criteria as a toxicity characteristic.
Inorganic Analysis
The results for the metals analyses of the arsenic-contaminated groundwater and
the EP extracts of the 17 wastes are presented in Table 14. These data are rounded off to
the nearest jjg/l except those for Hg (lower Standard) to facilitate comparison with RCRA
regulations. In comparison with the results of the organic analyses, these data suggest
that the EP extracts are predominantly ionic in character, as would be expected of
compounds extracted by an aqueous acidic extraction fluid. By the criterion of a
maximum permissible metal concentration in the EP extracts of 10 times the EPA Interim
Primary Drinking Water Standards (Table 12), three wastes (metal processing waste,
plater's waste, and municipal sewage sludge) plus the arsenic-contaminated groundwater
sample would be labeled by RCRA as "toxic" because of their extractable As, Cd, or Cr
contents, and one (power plant No. 1 fly ash) would be a borderline case due to its
extractable Cd content. However, a power plant fly ash sample from a second source,
as well as bottom ash, scrubber sludge, and treated scrubber sludge, contained far less
extractable Cd. None of the other energy-associated wastes (e.g., gasification wastes,
fluidized bed residue, shale) would receive a toxic classification by these criteria.
If EPA decided to expand the proposed toxicity characteristic (43 FR 58956) to
include the EPA Secondary Drinking Water Standards, then the dye waste also would be
classified as "toxic" because of its extractable Cu content. The extractable Zn and Cu
content of the plater's waste and the Zn content of municipal sewage sludge would provide
additional evidence for their "toxic" classifications. The extractable fluoride content of
the municipal sewage sludge would be a borderline classification. A measurement of
cyanide in the plater's waste EP extract revealed a 156 mg/l concentration. From these
results, the plater's waste and the waste contributing to the arsenic-contaminated
groundwater sample would be potentially the most "toxic" waste materials of the 18
examined.
36
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The increasing concentration trend for the metals Be, Cd, Cu, Ni, Pb, Sb, Tl,
and Zn in bottom ash < scrubber sludge < fly ash is interesting to note. The result is
consistent with the preferential accumulation of many of these elements in fly ash versus
bottom ash. However, other volatile metals, such as Hg, do not show the expected
concentration trends in the EP extracts.
A white precipitate formed in the EP extract of power plant No. 1 scrubber sludge
shortly after it was generated. An optical emission spectroscopic survey analysis
indicated that the major element was Ca, as would be expected from the limestone
employed in the scrubber. Approximately 0.1 mg/g of Mg and B also were detected in the
precipitate.
Although the RCRA protocol does not specify EP extract blanks, extraction blanks
were generated and analyzed to check the background levels of inorganic constituents.
Table 15 shows the analytical results for multiple blanks generated by the three types of
apparatus: A Plexiglas container with steel stirrer, a stainless steel container with
stainless steel stirrer blades, and a stainless steel container with Teflon stirrer blades.
There appear to be small differences among the blanks generated in the three apparatus;
notably between the Plexiglas and stainless steel apparatus. Because the former was
operated uncovered, the high result for one Zn determination may have been caused by
airborne contamination of the extraction vessel. The Plexiglas blank is appropriate for
the EP extracts of power plant No. 1 fly ash, bottom ash, and scrubber sludge;
gasification wastes No. 1-3; soybean process cake; metal processing waste; and plater's
waste. The stainless steel apparatus with the Teflon blades was used for the fluidized bed
residue, municipal sewage sludge, and power plant No. 2 fly ash EP extracts. The
stainless steel apparatus with the stainless steel blades was used for all the other EP
extracts. Although the magnitudes of the blanks are small in comparison with the
maximum permissible concentrations of metals, they are significant with respect to the
metals concentrations in some of the EP extracts, and they may decide a borderline case
such as the Cd level in the power plant No. 1 fly ash EP extract. Thus we feel that
appropriate EP extract blanks should be included in the RCRA protocol to aid in
classification of borderline cases.
ALTERNATE EXTRACTION PROCEDURES
The apparent failure of the EP protocol to produce extracts whose organic contents
mirror that expected to occur in a disposal environment has spurred the search for a more
meaningful extraction regimen with respect to organic pollutants. Several factors were
considered. The procedure should be (1) somewhat relevant to the actual (or supposed)
environmental extraction of these materials; (2) directly applicable to solid wastes
without physical modification of the waste (and, we hope, without regard for water
content); (3) amenable to routine analysis, i.e., rapid, facile, and quantitatively
accurate.
38
-------
TABLE 15. METALS ANALYSES OF EP EXTRACT BLANKS IN
THREE TYPES OF APPARATUS
Average concentration ± S.D. (jjg/l) for apparatus
Element Plexiglas Stainless steel Stainless steel
Ag
As
Ba
Be
Cd
Cr
Cu
Hg
Ni
Pb
Sb
Se
Tl
Zn
F
*
n = 4.
t
-------
The apparatus used in steam distillation (Figure 8) is commercially available
(Kontes Glass Co., Vineland, N. J., Part No. K523010). Briefly, the solid waste
(100 g) is added to a flask containing 250 ml of triply distilled water (represented by the
dark-colored fluid in Figure 8). The water is brought to a boil to accomplish steam
distillation. Concurrently, an immiscible organic solvent (represented by the light-
colored fluid in Figure 8) is distilled on the opposite side of the vessel. The steam and
organic vapors co-condense, and the two phases are separated and returned to their
respective vessels. Thus, an extraction is carried out in which the organic solvent does
not come into contact with the solid waste.
Pilot studies with this procedure have been promising. The recovery of
benzanthracene from water is virtually 100%, whereas higher-boiling pollutants such as
benzo(a)pyrene are only partially recovered. Furthermore, the recovery depends on the
nature of the solid waste.
Recovery of the benzanthracene tracer from the metal processing waste was 60%,
whereas recovery from the soybean process cake was essentially 0. Recovery is a measure
of the adsorptivity of the solid, and adsorptivity probably affects the release of organ!cs
into the environment from a landfill disposal site.
Two of the solid waste samples that were subjected to the EP and subsequent
biological, analytical, and ecological testing were chosen for evaluation based on this
technique; the two samples, metal processing waste and soybean process cake, were
selected based on their substantial physical differences, one being a clay and the other
resembling activated charcoal. Neither of these materials showed significant biological
activity when tested by the proposed RCRA procedure. 4C-labeled benz(a)anthracene
was used for recovery measurements. GC on an OV-101 glass capillary column was used
for preliminary screening of the total extract as well as for analysis of the fractions after
isolation.
GC analysis of the two steam distillation extracts followed the pattern expected
from the tracer recoveries. The metal processing waste was considerably more complex
in organic content than the soybean process cake (Figures 9 and 10). The extract of the
former, representing 0.2% by weight of the solid, was fractionated to obtain an isolate
containing PAHs. Fluoranthene, pyrene, benz(a)anthracene, chrysene, and
benzo(b)fluoranthene were tentatively identified~by their GC retention times. Again, no
PAHs were found in the EP extract.
The steam distillate extracts were also subjected to mutagenicity testing in the
Ames/Salmonella system (see Sect. 8). The metal processing waste again showed
significant activity, but the soybean process cake was inactive. Thus, this extraction
procedure offers a substantial improvement over the EP/ at least with respect to recovery
of organic pollutants. An extraction procedure such as this, effective for organic
compounds, should be incorporated into the RCRA protocol.
40
-------
2445-79
Figure 8. Steam distillation apparatus for solid waste extraction,
-------
ORNL-DWG 79-12306
STEAM DISTILLATE OF METAL WASTE
30
45
TIME (min)
60
75
Figure 9. Glass capillary column GC separation of steam distillate from
metal processing waste.
STEAM DISTILLATE OF SOYBEAN PROCESS CAKE
jaLiOlwA^
ORNL-DWGr9-<2307
15
30
45
TIME ( min )
60
75
Figure 10. Glass capillary column GC separation of steam distillate from
soybean process cake.
42
-------
CONCLUSIONS AND RECOMMENDATIONS
The overriding conclusion from the chemical analyses is that the current EP
protocol is ineffective in extracting organic compounds from solid wastes. The ionic
nature of the extractant fluid in the current EP/ the open system extraction, and possibly
the membrane filtration step work against extraction and recovery of organics. As would
be expected, the EP extracts generated by the current EP are predominantly ionic in
character, and only the dissolved metals analyses are useful for determination of the
"toxic"/"nontoxic" classification of the solid waste. It probably is not necessary to
conduct organic compound analyses of the extracts generated by the current EP/ although
a relatively inexpensive screening procedure such as that for total organic carbon might be
useful for setting priorities for organic analyses if acetic acid was not included in the EP.
Alternate extraction procedures may be necessary for assessment of ex tractable
organic compounds. One possible candidate is the continuous steam distillation/organic
solvent extraction method examined in our pilot study.
Research should be conducted into the physical/chemical properties of wastes which
dictate availability of organic/inorganic constituents and on means of cost-effective
determination of these properties in the wastes. When available, authentic landfill
leachates should be compared with extracts prepared in the laboratory to assess the
environmental relevance of an extraction procedure. The ideal extraction procedure
would reproduce the composition of an actual leachate. Direct analysis of the solid
waste also should be considered.
43
-------
SECTION 6
AQUATIC TOXICITY
The purposes of the aquatic toxicity tests were: (1) to evaluate a short-iterm
screening test for its suitability for determining if an EP extract poses a hazard to the
aquatic ecosystem under conditions described in EPA's draft RCRA 83001 Toxicity
Background Document, and (2) to determine the toxicity of the arsenic-contaminated
groundwater and 17 waste extracts to aquatic organisms. Lethal or acute (short-term
exposure) tests and chronic (long-term) tests, which determined sublethal effects on
reproduction, were used. The test organism was the cladoceran, Daphia magna, which is
used as a standard organism in toxicity testing because of its sensitivity to many chemicals.
From the results of the acute tests, the concentration of the extract that was lethal to D.
magna during an exposure period of 48 h was estimated. From the chronic test results, it
was determined if continuous exposure to extract dilutions of 1:100 and 1:1000 impaired
reproduction of D. magna. The chronic tests did not, therefore, establish a dose-response
relationship, such as an LC5Q, for the extract but only determined if the extract was
toxic at the concentrations tested. The 1:1000 dilution factor was proposed by EPA as
being a reasonable dilution factor for protection of aquatic organisms from chronic
exposure to leachates from landfill waste.
The test procedures are described in Appendix D. The five criteria used to assess
the effects on D. magna reproduction in the chronic tests were survival time, day of first
brood release, number of broods per adult, number of young per brood, and total number
of young produced per adult. From the results of the chronic exposure tests, the most
consistently sensitive criteria were determined for evaluation of toxic effects. The
statistical tests used to compare the sensitivities of the above criteria were analysis of
variance combined with Duncan's Multiple Range Test to identify significant differences
among individual treatment means. A type I error (oj of 0.05 was selected as the level
of significance.
RESULTS
The results of the acute toxicity tests are shown in Table 16, and those of the
chronic tests are presented in Table 17. A summary of the results for both types of tests
and their significance is given in Table 18. The second acute toxicity test was done
28 days after the first one to determine if the toxicity of the test material had changed
44
-------
TABLE 16. ACUTE TOXICITY OF WASTE EXTRACTS TO D. MAGNA
48-h LC5Q* (95% fiducial limits)
Extract
Arsenic-contaminated groundwater
Soybean process cake
Metal processing waste
Plater's waste
Raw shale
Retorted shale
Dye waste
Textile waste
Municipal sewage sludge
Power plant No. ? fly ash
Power plant No. 1 bottom ash
Power plant No. 1 scrubber sludge
Power plant No. J treated scrubber sludge
Power plant No. 2 fly ash
Fluid! zed bed residue
Gasification waste No. 1
Gasification waste No. 2
Gasification waste No. 3
First test
2.2*
*
28.01"
0.005
53.9(47.1-62.3)
70.0 (30.0->100)
o.ooosf
19.2 (16.8-22.5)
7.5t
90 (71->100)
94t
85 (51-»100)
81.1 (72.8-91.0)
15.2t
12.6 (10.8-15.0)
§
100. 0*
70.0 (29.4->100)
Second test
1.7*
§
39.01"
not done
64.4 (56.
39.4 (30.
not done
35. 5t
3.0*
69t
60t
60t
95. 6 (85.
11.6*
25.0
§
*
46.0*
1-77.6)
7-51.4)
2->100)
*Est?mated percent concentration of the extract that killed or immobilized 50% of the
test organisms in 48 h.
'Approximate value; 95% fiducial limits could not be calculated.
*No significant mortality of test organisms in 48 h in 100% of the extract.
§ Less than 50% of the test organisms were killed or immobilized in 100% of the extract
?n48h.
during that time, which was the duration of the chronic toxicity tests.
stored at 4°C during the 28 days.
Extracts were
A material was considered to have caused a toxic effect when there was a
statistically significant reduction (a = 0.05) in the mean number of young produced by
each organism at either test dilution compared with the number of young produced by
control organisms exposed to the dilution water alone and dilution water with acetic or
hydrochloric acid. Of the above-mentioned five criteria used to assess toxicity, the
mean number of young per adult and the number of young per brood were the most
consistently sensitive; the first was chosen for convenience as the toxicity criterion.
45
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50
-------
TABLE 18. SUMMARY OF THE D. MAGNA ACUTE AND
CHRONIC TOXICITY~TEST RESULTS
Significance* of chronic
toxicity test results
at dilution
Extract
(% cone.)
1:1000
1:100
Arsenic-contaminated groundwater
Soybean process cake
Metal processing waste
Plater's waste
Raw shale
Retorted shale
Dye waste
Textile waste
Municipal sewage sludge
Power plant No. 1 fly ash
Power plant No. 1 bottom ash
Power plant No. 1 scrubber sludge
Power plant No. 1 treated scrubber sludge
Power plant No. 2 fly ash
Fluidized bed residue
Gasification waste No. 1
Gasification waste No. 2
Gasification waste No. 3
2.2
No significant
mortality
28.0
0.005
53.9
70.0
0.0005
19.2
7.5
90
94
85
81.1
15.2
12.6
<50% mortality
100.0
70.0
NS
NS
NS1"
S
NS
NS
S
NS
S
NS
NS
NS1"
NS*
NS
NS
NS
S
NS
S
NS
NS*
S
NS
NS
S
NS
S
NS
NS
NS1"
NS
NS
NS
NS
NS
NS
The criterion for significance was a reduction (a= 0.05) in the mean number of young
produced at either test dilution compared with the dilution water and acetic acid
controls. NS, not significant; S, significant.
"Not significant when the treatment mean was compared with the dilution water control
mean, but significant when compared with the acetic acid control mean.
*Not significant when the treatment mean was compared with the acetic acid control
mean, but significant when compared with the dilution water control mean.
51
-------
Arsenic-Contaminated Groundwater
This material was toxic to D. mag no in both acute and chronic tests. The 48-h
LC5Q concentrations were 2.2 and 1.7% (Table 16). In the chronic toxicity tests there
were no significant effects on reproduction or survival at the 1:1000 dilution, but at the
1:100 dilution significant effects were found by three of the five toxicity criteria
(Table 17). The mean number of young produced per adult and the mean number of
young per brood were both reduced by 88% compared with the dilution water controls.
Also, the mean day of first brood release significantly increased from 11.3 for the
controls to 13. 7 for animals in the 1:100 dilution.
Soybean Process Cake
In the first acute toxicity test, no animals were killed or immobilized after an
exposure to 100% of the extract for 48 h; in the second test less than 50% were adversely
affected (Table 16). Results of the chronic toxicity test also showed the low toxicity of
the extract for D. mag no (Table 17). There were no significant reductions in any of the
toxicity values obtained for the test animals compared with the control values at either
of the two test dilutions.
Metal Processing Waste
This material was toxic to D. mag no in the acute toxicity tests with 48-h LC5QS
of 28.0 and 39.0% (Table 16), but it was not toxic at the test dilutions used in the
chronic toxicity tests (Table 17).
Plater's Waste
The acute toxicity test done with this material showed that it was highly toxic to
D. magna. The 48-h LC^g was 0.005% (Table 16). Because this value is less than the
0. 1% dilution used in the chronic toxicity tests, these tests were not done.
Raw Shale
This material had low toxicity to D. magna in the acute toxicity tests. The
48-h LC5QS were 53.9 and 64.4% (Table 16). By three of the five criteria used in the
chronic toxicity tests, there were no effects of the material on the organisms (Table 17).
With mean number of young per adult and mean number of young per brood as criteria,
there is evidence for stimulation of reproduction at 1:100 dilution and no or little
inhibition at 1:1000 dilution (Table 17).
Retorted Shale
As was the case with raw shale, retorted shale had low toxicity for D. magna in
the acute toxicity tests. The 48-h LC50s were 70.0 and 39.4% (Table 16)7 The
52
-------
effects in the chronic toxicity tests were not significantly different from the control
values (Table 17).
Dye Waste
This material was very toxic to D. mag no. The 48-h LCjQ was 0.0005%
(Table 16). Because this value is less than the 0. 1% dilution used in the chronic
toxicity tests, these tests were not done.
Textile Waste
This material was moderately toxic to D. mag no in the acute toxicity tests. The
48-h LCggs were 19.2 and 35.5% (Table 16). In the chronic toxicity tests, there were
no effects by three of the five toxicity criteria (Table 17). By the other criteria,
reproduction was stimulated at the 1:100 dilution but the 1:1000 dilution had no effect.
Municipal Sewage Sludge
This material was toxic to D. magna in both the acute and chronic toxicity tests
(Tables 16 and 17). The 48-h LCjQs were 7.5 and 3.0%.
Power Plant No. 1 Fly Ash, Bottom Ash, and Scrubber Sludge
These materials had low toxicity to D. magna in the acute toxicity tests
(Table 16). The 48-h LC^QS for the fly ash, bottom ash, and scrubber sludge were 90
and 69%, 94 and 60%, and 85 and 60%, respectively. They also had no effect in the
chronic toxicity tests (Table 17).
Power Plant No. 1 Treated Scrubber Sludge
This material had low toxicity to D. magna in the acute toxicity tests. The 48-h
LC«jgs were 81. 1 and 95.6% (Table 16). In the chronic toxicity tests, there was no
effect by either the 1:100 or 1:1000 dilution on D. magna reproduction (Table 17) when
compared with the acetic acid control. However, a difference was noted between the
dilution water control and the 1:1000 dilution.
Power Plant No. 2 Fly Ash
In the acute toxicity tests, this material showed a moderate toxicity to D. magna.
The 48-h LC5QS were 15.2 and 11.6% (Table 16). This material was not toxic at either
test dilution in the chronic assay, however (Table 17).
53
-------
Fluidized Bed Residue
This material was moderately toxic to D. magna in the acute toxicity tests. The
48-h LC^QS were 12.6 and 25.0% (Table 16). The material was not toxic at either test
dilution in the chronic toxicity tests (Table 17).
Gasification Wastes No. 1—3
These materials had low toxicities to D. magna in the acute toxicity tests (Table
17). Toxic effects occurred only with full or near full strength solutions. Results of the
chronic toxicity tests (Table 17) showed no effects of these wastes except for the 1:1000
dilution of waste No. 2 (Table 17). But since there were no significant effects with this
material at 1:100 dilution, the apparent effects with the higher dilution become less
important in evaluating the toxicity of waste No. 2 than if significant effects were found
for both dilutions.
DISCUSSION
In these chronic toxicity tests, the effects of the test materials on D. magna were
determined by the use of five population characteristics as toxicity criteria. Of these
criteria, the most consistently sensitive were the number of young per brood and the
number of young per adult; survival time, the day of first brood release, and the number
of broods produced were affected less by the extracts. Since the total number of young
produced per adult is in part a function of the number of young per brood, the former was
chosen for convenience to assess toxicity. Results from these studies and from other
studies with the 28-day chronic toxicity test with D. magna support the following
conclusions:
(1) The mean number of young produced per brood and the mean total
number of young produced by each female are the most consistently
sensitive population characteristics of those tested to chemical
toxicity.
(2) With these two characteristics as toxicity criteria, the test can be
used reliably to determine the maximum concentration of a chemical
that produces no significant observed toxic effects and the minimum
concentration that produces such effects. To do so, however, requires
more than the two test concentrations used in the present screening
tests.
The criterion selected to identify extracts as toxic to D. magna and as potentially
hazardous to aquatic organisms was a significant reduction (a= 0.05) in the mean number
of young produced by each D. magna during the 28-day test period compared with the
number produced by organisms exposed to the dilution water and acid controls. By this
criterion, 4 of the 18 tested extracts, namely, arsenic-contaminated groundwater,
plater's waste, dye waste, and municipal sewage sludge, would be classified as potentially
54
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hazardous (Table 18). Of the remaining extracts, only the metal processing waste and
power plant No. 1 untreated and treated scrubber sludge showed any significant effects,
but only when the results with the two test dilutions were compared with either the acetic
acid controls or dilution water controls (Table 18).
On the basis of screening test results, it is not possible to definitely determine if
waste would be hazardous to a natural population or community of aquatic organisms.
There are limitations with the extrapolation of laboratory results based on single species
tests to the prediction of effects on natural ecosystems. The single-species test systems
are simple compared with complex natural ecosystems. Thus, even though a significant
toxic effect can be demonstrated in the laboratory, under natural conditions it may be
mitigated, modified, or even magnified by the chemical, physical, and biological
interactions of the ecosystem. However, D. magna is sensitive to many classes of toxic
chemicals, and thus results of toxicity tests with this species can be regarded as
conservative predictors of potential hazard to other aquatic species.
Because of limitations with the EP used for the D. magna chronic toxicity tests,
some toxic wastes may not have been identified. For example, all volatile chemicals
were probably lost during the extraction process. Also, the renewal-type test procedure
used in the chronic toxicity tests is not as efficient as continuous flow-through test
systems for detecting nonpersistent chemicals.
In summary, the chronic test results can be used to identify only those wastes that
are potentially hazardous. More in-depth tests under site-specific conditions with
several test organisms would be necessary to determine conclusively if a waste was
hazardous to a particular aquatic ecosystem.
Comparison of results from the second set of acute toxicity tests, which were done
28 days after the first ones, with results from the first set determined that the toxicities
of some extracts increased during storage, whereas those of others decreased (Table 16),
but the differences are probably not statistically significant for arsenic-contaminated
groundwater, soybean process cake, and metal processing waste. Data for the remaining
extracts are insufficient for statistical analysis.
RECOMMENDATIONS
The 28-day chronic toxicity test with D. magna can detect toxic materials in
extracts (Table 18). However, reproduction of D. mag no is affected by food supply.
Some neutralized test extracts contained high concentrations of acetate, which can
stimulate bacterial reproduction. Bacteria are food for D. magna. Acetate can serve
as food for bacteria, and this increased bacterial level could indirectly stimulate D.
magna reproduction. Of the 15 tests in which both an acetate and dilution wateF
control were used, 12 showed no significant differences in reproduction and 3 showed
slightly higher fecundity in the acetate controls relative to dilution water controls.
These results may be explained by the concentration of acetate in the water. Results of
55
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some of our other research suggest that low concentrations of acetate stimulate
reproduction, but that concentrations ^0. 1% are toxic. Nevertheless, interactions of
acetate with other toxic chemicals in the waste materials are not known. These
interactions could have synergistic or antagonistic toxic effects on aquatic organisms,
which could make the interpretation of the test results more difficult. Therefore,
additional work should be conducted to determine the effects acetic acid has on aquatic
organisms, and, if necessary, an EP to determine aquatic toxicity should be developed to
correct this problem.
REPRODUCIBILITY OF CHRONIC TOXICITY TEST
There is little information on the reproducibility of results obtained with the 28-day
D. mag no chronic toxicity test, which was the test used in the above screening
experiments. Therefore, we did a study to determine if this chronic toxicity test will
yield reproducible results.
We chose the azaarene, acridine, as the test material because of our experience
with it in testing coal conversion products and wastes. The materials and methods were
those used in the above screening tests (Appendix D), except that a series of test dilutions
were used instead of just two, and 20 animals were used for each test concentration
instead of 10. In this study, "no observed effects" concentrations (NOECs) were
determined, which are defined as the highest concentration of a chemical that causes no
statistically significant observed effects on the test organisms compared with the controls.
The NOECs ranged between 0.4 and 1.6 mg/l of acridine in the four tests (Table
19). The NOECs for survival, age at onset of reproduction, and number of broods per
TABLE 19. NOECs FROM FOUR TESTS ON THE CHRONIC TOXICITY
OF ACRIDINE TO D. MAGNA
NOEC (mg/l) for test No.
Toxicity criterion
Survival
Occurrence of pri mi porous instar
Age at onset of reproduction
No. of broods/female
No. of young/brood
No. of young/female
0.8
0.8
0.8
0.8
0.4
0.4
1.6
0.8
0.8
0.4
0.4
0.4
0.4
0.8
0.4
0.4
0.4
0.4
0.8
0.8
0.4
0.8
0.4
0.4
56
-------
female varied significantly among the four tests, but the NOECs for occurrence of
primiparous instar (when young are first present in the female's brood chamber), number of
young per brood, and number of young per female did not vary at all in the four tests.
The latter two criteria were the most sensitive for estimation of the NOEC, since their
NOEC (0.4 mg/l) was less than the value for occurrence of the primiparous instar
(0.8mg/l).
The responses of D. magna to various acridine concentrations were usually not the
same among the four tests (Table 20). At 3.2 mg/l the results were identical among the
tests for five of the six toxicity criteria, but at this concentration all means were zero
since reproduction was completely inhibited by acridine. Results of the study by Canton
and Adema'2 on tne reproducibility of a 21-day D. magna chronic toxicity test appear to
support our findings. Variations in the inhibition of reproduction were 20% or greater in
their duplicate tests with six compounds; however, NOECs appeared to be reproducible
in most of their experiments. Our interpretation of their results is tentative since
statistical analyses were not provided.
In conclusion, the D. magna static-renewal chronic toxicity test using either the
number of young per brood or number of young per female as the toxicity criterion is a
reproducible and sensitive test.
TABLE 20. VARIABILITY IN RESPONSE OF D. MAG NATO ACRIDINE
IN FOUR CHRONIC, STATIC-RENEWAL TESTS*
Significance of variation at acridine
concentration (mg/l)
Toxicity criterion
Survival
Occurrence of primiparous
instar
Age at onset of reproduction
No. of broods/female
No. of young/brood
No. of young/female
0.0
(control)
NS
S
S
S
S
S
0.0
(methanol
control)
NS
S
S
S
S
S
0.2
NS
S
S
S
S
S
0.4
S
S
S
S
S
S
0.8
S
S
S
S
S
S
1.6
S
S
S
S
S
S
3.2
S
NS
NS
NS
NS
NS
CNS, not significant at a = 0.05; S, significant.
57
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SECTION 7
PHYTOTOXICITY
PROBLEM DERNITION
The problem posed to us by EPA was to develop and evaluate short-term screening
bioassay tests for determining whether materials extracted from a waste, as determined by
the EPf would be harmful to terrestrial plants if the leachate was used as irrigation water.
Toward this end short-term root elongation and seedling growth assays were done on 17
extracts and one groundwater sample. Problems were anticipated because of reports in
the literature that acetic acid/acetate ion, even at relatively low levels, can be
phytotoxic.13
METHODOLOGY
Short-term (48- and 72-h) root (radicle) elongation tests were performed with
radish and sorghum in a controlled environment. Longer-term (2- and 3-week) seedling
growth studies were carried out in pots under greenhouse conditions with wheat and
soybean. Parameters measured were root length in the short-term tests and root and shoot
dry weight (or length in some cases) in the longer-term tests. Treated plants were
compared with plants grown in distilled water in the case of the short-term tests or a plant
nutrient solution in the longer-term study. Treated plants received a 10% concentration
of the EP extract (diluted with nutrient solution) in most of the long-term tests to simulate
a tenfold dilution that EPA assumes will occur as water drains from a natural landfill to
groundwater. This groundwater could then be used to irrigate crops. However, following
seedling growth studies with acetic acid, we decided to dilute the fluidized bed residue
and the power plant No. 1 treated scrubber sludge samples to 2 and 8%, respectively, to
reduce phytotoxic effects of acetic acid. In the short-term tests, treated plants received
a series of concentrations of the EP extracts. Preliminary results from radish and sorghum
treated with different concentrations of acetic acid showed inhibited plant growth only
when the acetic acid concentrations exceeded —5 ml of 0.5 N acetic acid per I (2.5
mea/l). The highest concentration to be tested was chosen by diluting the volume of
acetic acid added during extraction to approximately the 5 ml/I toxicity threshold.
The plant species selected for these tests are important agriculturally and represent
the two major classes (monocotyledons and dicotyledons) of flowering plants. Because of
the difference in growth habits of these two classes of plants, they may respond differently
58
-------
to various chemicals in their environment. Of course, this is also true to a lesser extent
between individual species of the same class, but it was not feasible to test large numbers
of many different species. More detailed methods are given in Appendices E and F.
RESULTS AND DISCUSSION
Arsenic-contaminated groundwater was highly phytotoxic, showing 33% growth
reduction of radish roots (radicles) even at a 2% concentration. Higher concentrations
(10 and 25%) reduced growth 70 and 59%, respectively. However, at the highest
dilution (0.1%) there was a slight stimulation of root growth.
Data from other tests in which extracts were obtained by the EPA EP are presented
in Tables 21 and 22. Table 23 presents data on acetic acid effects (see "Problem Areas").
Table 24 is a summary of the results presented in these three tables.
Of the coal processing wastes, three (power plant No. 2 fly ash, gasification
waste No. 1, and fluidized bed residue) were not toxic. Retorted shale and power plant
No. 1 treated scrubber sludge reduced growth of the monocotyledon species (sorghum and
wheat) in both types of tests. The latter inhibited both root and shoot growth of wheat;
the former inhibited only root growth. Raw shale caused a growth reduction of 14% in
sorghum roots. These three extracts were phytotoxic at less than a tenfold dilution.
Power plant No. 1 scrubber sludge was toxic to radish seeds in the root elongation
test even at a 10% concentration, but the same concentration was not toxic to sorghum.
Power plant No. 1 bottom ash and fly ash were only slightly toxic to either radish or
sorghum at concentrations exceeding 10%. In the seedling growth studies power plant
No. 1 fly ash caused a slight but significant reduction of wheat root weight but not shoot
weight.
Results of the other two gasification wastes showed the waste designated No. 2 to
be the only one of the three which was toxic at a concentration less than 10%. This
effect was seen in the seedling growth study with soybean roots. Gasification waste
No. 3 caused 22 and 12% growth reductions of radish and sorghum roots, respectively, at
100% concentration.
Municipal sewage sludge was the only one of six industrial wastes that was not
toxic. Soybean process cake significantly inhibited root growth of wheat and sorghum.
Although the latter monocotyledon species showed no effect at 50% concentration after
72 h, the waste was toxic to wheat exposed for 2 weeks even though the solution was more
dilute (10% concentration). Metal processing waste also affected monocotyledon root
growth significantly.
59
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TABLE 21 . RADISH AND SORGHUM RADIC1.F LENGTHS*
Extract
Arsenic -contaminated
groundwater
Soybean process cake
Metal processing waste
Plater's waste
Raw shale
Retorted shale
Concentration
10
5
2
0.1
control
75
control
75
50
control
4
control
4
control
5
2
control
0.5
control
5
control
4
control
4
2
control
2
control
2
1
control
0.5
control
Seed
type
radish
radish
sorghum
radish
sorghum
radish
sorghum
radish
sorghum
radish
sorghum
Root length1
(mm)
8± 6
11 ±7
18 ±8
31 ± 12
27± 14
23 ± 10
22 ± 10
23 ± 10
28 ± 14
27± 12
27 ± 13
28 ± 13
29 ± 13
33 ± 13
14 ± 6
22 ± 8
28 ± 13
33 ± 12
31 ± 14
12 ±5
11±5
23 ± 12
24 ± 12
25 ± 11
29 ± 12
29 ± 12
25 ± 11
25 ± 13
25 ± 11
25 ± 11
29 ± 12
42 ± 18
38 ± 18
% Growth
reduction
70*
33*
0
—
0
15*
0
—
4
12*
50*
21*
—
0
—
0
4
—
14*
0
—
0
—
14*
14*
—
0
—
(continued)
60
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(Table 21 continued)
Extract
Dye waste
Textile waste
Municipal sewage sludge
Power plant No. 1 fly ash
Power plant No. 1
bottom ash
Power plant No. 1
scrubber sludge
Concentration
(%)
10
5
control
10
5
control
0.5
control
5
2
control
5
control
20
control
20
control
30
control
30
control
100
control
100
control
10
control
10
control
Seed
type
radish
sorghum
radish
sorghum
radish
sorghum
radish
sorghum
radish
sorghum
radish
sorghum
Root length t
(mm)
21± 11
23 ± 10
25 ± 13
9±5
9±4
11±5
41 ± 17
38 ± 16
22 ±9
27 ± 11
28 ± 13
30 ± 11
33 ± 13
35 ± 12
33 ± 12
28 ± 9
29 ± 12
27± 15
31 ± 16
15 ± 13
I7± 15
21 ± 11
22 ± 12
20 ± 12
23 ± 13
16 ± 11
20 ± 15
38 ± 30
39 ±28
% Growth
reduction
16*
8
18*
18*
0
21*
4
9*
0
3
13*
12
5
13*
20*
3
(continued)
61
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(Table 21 continued)
Extract
Power plant No. 1 treated
scrubber sludge
Power plant No. 2 fly ash
Fluldized bed residue
Gasification waste No. 1
Gasification waste No. 2
Gasification waste No. 3
Concentration
(%)
8
control
8
4
control
100
control
100
control
2
control
2
control
100
control
100
control
100
control
100
control
100
50
control
100
control
Seed
type
radish
sorghum
radish
sorghum
radish
sorghum
radish
sorghum
radish
sorghum
radish
sorghum
Root length'''
(mm)
32 ± 10
34 ± 12
30 ± 10
33 ± 12
33 ± 13
32 ± 10
34 ± 12
31 ± 11
32 ± 11
34 ± 13
36 ± 15
40 ± 14
42 ± 16
21 ± 10
21 ± 12
21 ± 11
23 ± 16
27± 12
25 ± 12
24 ± 13
19± 13
24 ± 12
32 ± 15
31 ± 17
22 ± 11
25 ± 9
% Growth
reduction
6
9*
0
6
3
6
4
0
9
0
0
22*
0
12*
*Radish and sorghum were grown at 25°C in the dark for 48 and 72 h, respectively.
Controls were grown with distilled water and compared with plants treated with the
indicated concentration of extract.
'Values given are mean ± S.D.
Significantly different from controls at a 5% probability level.
62
-------
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