United States
Environmental Protection
Agency
Environmental Monitoring Systems"^"
Laboratory
Las Vegas NV89114
Research and Development
EPA-600/S4-83-001 Mar. 1983
SERA Project Summary
Mobility of Organic Compounds
from Hazardous Wastes
D. K. Brown, M. P. Maskarinec, F. W. Larimer, and C. W. Francis
The objective of this research is to
develop a second generation laboratory
extraction test to model the mobility of
organic and inorganic constituents
from solid wastes co-disposed with
municipal waste. This test should more
accurately and reproducibly model
leachate production, for selected
organic as well as inorganic constitu-
ents, than the test procedure referred to
as EP promulgated by EPA in 1980.
As a first approach the capabilities of
five aqueous extraction procedures to
remove organic compounds from 11
solid wastes were evaluated. The
extraction procedures investigated
were four batch extractions using (1)
deionized distilled water adjusted to pH
5 with 0.5 N acetic acid,|(2) deionized
distilled water, (3) deionized distilled
water with a sodium cation exchange
resin, (4) 0.5 M sodium citrate, and (5)
an upward-flow column extraction
using deionized distilled water. The
major conclusions relative to the
effectiveness of the extraction
procedures to remove organic
compounds were: (1) the column
procedure extracted more organic
material than any of the batch proce-
dures, and (2) among the batch
extraction procedures, deionized
distilled water was the most aggressive
medium.
The most noticeable differences
between the column procedure utilized
and the batch procedures were the
elevated levels of moderately volatile
and the nonpolar organic compounds
found in the column extracts. Factors
contributing to these results are: (1) the
column procedure is a completely
closed extraction permitting direct
collection of volatile compounds, and
(2) the column extracts were not
filtered through membrane filters
which are known to sorb appreciable
quantities of nonpolar compounds.
Two extracting devices (magnetically
stirred and rotary extractor) for
conducting the EP were also compared.
Extracts produced by the two
extractors showed significantly
different concentrations of As, Cd, Fe,
Ni, and Zn, although neither method
showed a consistent pattern.
The proposed reverse-phase High-
Pressure Liquid Chromatography
protocol to assess the bioaccumulation
potential of solid waste extracts was
found to produce only qualitative
information because of nonuniformity
of detector response. However, the test
does provide a useful screening method
for the detection of potentially bioac-
cumulative organic compounds.
In addressing a secondary research
objective, comparison of two isolation
techniques (i.e., resin adsorption
technique using Amberlite XAD-2 resin
and a solvent partition technique using
methylene chloride) to isolate organic
mutagens from aqueous solutions for
testing in the Ames Salmonella
mutagenicity assay was conducted.
Although the assay results were not
affected by the type of isolation
technique used, the extraction
efficiency of the resin technique was, in
general, less dependent on the specific
agueous medium than was the solvent
partition.
This Project Summary was developed
by EPA's Environmental Monitoring
Systems Laboratory. Las Vegas. NV, to
announce key findings of the research
project that is fully documented in a
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separate report of the same title (see
Project Report ordering information at
back}.
Introduction
Under Section 3001 of the Resource
Conservation and Recovery Act of 1976
(RCRA), the U.S. Environmental Protec-
tion Agency (EPA is charged with
identifying industrial wastes which, if
improperly managed, pose a potential
hazard to human health or the
environment. To carry out this mandate,
EPA identified a number of properties
exhibited by a waste which would
indicate that the waste requires
controlled management. One of these
properties relates to the degree to which
toxic species might leach out of a waste
and contaminate groundwater if the
waste were disposed of in a nonsecure
municipal landfill. The toxicity test proce-
dure promulgated by EPA (USEPA 1980a)
for use in determining if an unacceptably
high potential for groundwater contamin-
ation might occur is called the Extraction
Procedure (EP).
The EP produces an extract that is
analyzed for the eight elements (As, Ba,
Cd, Cr, Pb, Hg, Se, and Ag), four pesticides
(Endrin, Lindane, Methoxychlor, and
Toxaphene), and two herbicides [2,4-D
and 2,4,5-TP (Silvex)*] for which National
Interim Primary Drinking Water
Standards (NIPDWS) (USEPA 1979) have
been established. A waste is defined as
hazardous if the EP extract of the waste
contains any constituent at a
concentration equal to or greater than
100 times the NIPDWS concentration.
These hazardous waste threshold levels
have been established taking into
account attenuation and dilution
processes expected to occur during the
movement of leachate through the
underlying strata and groundwater
aquifer.
The EP is considered to be a first-order
approximation which primarily models
the leaching action of the low molecular
weight carboxylic acids generated in an
actively decomposing municipal waste
landfill. In the EP, acetic acid is added to a
solid waste suspension in distilled water.
The acetic acid primarily affects the
leaching of metals from industrial waste.
The higher molecular weight organic
compounds present in municipal landfill
leachates that are expected to affect the
"Mention of trade names or commercial products
does not constitute endorsement or recommendation
for use by the U S Environmental Protection Agency
teachability of nonpolar organic com-
pounds are not currently modeled by the
EP. This perceived limitation is the impe-
tus behind the current research.
The objective of this research is to
develop a second generation extraction
test that will more accurately and
reproducibly model leachate production,
for organic as well as inorganic
constituents, in the previously described
disposal environment. A secondary
objective is that this test be amenable to
subsequent biological testing. Work
during this reporting period has centered
on four separate but related tasks:
Task 1: An evaluation of aqueous
extraction procedures to
remove nonpolar organic
compounds from solid
Task 2: Comparison of two sample
preparation protocols for per-
forming the Ames test on
solid waste extracts and
wastewaters.
Task 3: An evaluation of the equiva-
lence of a magnetically stirred
extractor relative to an EPA-
approved rotary extractor for
conducting the EP.
Task 4: An evaluation of the proposed
reverse-phase High-Pressure
Liquid Chromatography
(HPLC) protocol for assessing
the bioaccumulation poten-
tial of solid waste extracts.
Experimental
Task 1
The primary objective of Task 1 was to
assess the capabilities of five selected
extraction procedures to remove organic
compounds, determined by gas chroma-
tography (GC) and gas chromatography/
mass spectrometry (GC/MS) analyses,
from 11 solid wastes known to contain
significant quantities of organic
compounds.Other objectives included
examining the solid waste extracts for
selected inorganic constituents known to
be present in the wastes and
mutagenicity testing of the extracts.
Mutagenicity testing was included to
evaluate any difference in aggressive-
ness toward organic compounds
between extraction procedures that
might not be detected by GC analyses;
e.g., the ability of an extraction procedure
to extract nonvolatile mutagens from
solid wastes, and to determine the
compatibility of the extracts produced,
with the Ames assay procedure.
The five extraction procedures selected
for this study included four batch
extractions and an upward-flow column
extraction. Each procedure utilized a final
liquid to solid ratio of 20:1. The following
media were used:
Batch 1: Deionized distilled water with
the solid waste suspension
adjusted to pH 5 with 0.5 N
acetic acid (EP)
Batch 2: Deionized distilled water
Batch 3: Deionized distilled water with
a sodium cation exchange
resin
Batch 4: 0.5 M sodium citrate buffer
Column: Deionized distilled water
Eleven solid wastes from a variety of
industries were obtained and extracted
using the five different extraction
procedures. Table 1 contains a listing of
the extraction procedures with a compila-
tion of variable leaching factors.
The EP was performed according to
current regulatory protocol (USEPA
1980a). The water and Na-resin
extractions were performed in the same
manner as the EP, except for the change
in extraction media. The citrate buffer
extraction utilized a rotary extractor
rather than the magnetically stirred
extractor used with the other batch
extractions.
The column extraction utilized solid
waste thoroughly mixed 1:1 by weight
with acid-washed sea sand to increase
the hydraulic conductivity of the samples.
A glass column 2.5cm i.d. and45cm long
was packed with the solid waste/sand
mixture. A 2.5-cm layer of sand was put
on each end of the sample . Deionized
distilled water (passed through XAD-2
resin to remove trace organic material)
was pumped upward through the solid
waste until an effluent equivalent to a
1:20 solid:liquid ratio was obtained.
Effluent from the column was passed
through an in-line 10-//m pore size PTFE
cloth, located on top of the column, and
then directly through a 16.8-mL cartridge
of XAD-2 resin for collection of nonpolar
organic compounds.
The organic analysis of the various
batch aqueous extracts was carried out
as follows: the aqueous extract was
adjusted to ph 6.8 using a phosphate
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Table 1.
Extraction
Extraction Procedures: Identification of Variable Leaching Factors
Variable Factors*
Initial Leaching Medium Mode of Extraction
pH Adjustment
Treatment of Leachate Solution
for Extract Analysis
1. Batch 1: EP
Deionized distilled water** Batch: magnetically
stirred
2. Batch 2: Water Deionized distilled water** Batch: magnetically
stirred
3. Batch 3: Na-resin Deionized distilled water** Batch: magnetically
with 1 -g calculated dry wt stirred
Chelex 100/10-g sample
Adjust to pH 5 with 0.5 N
acetic acid - maximum limit
of 2-meq/g sample
None
Pressure-filtered through,
0.4-fjm Nuclepore filter
Pressure-filtered through
0 4-fjm Nuclepore filter
Adjust to pH 7 with 0.1 N HCI Pressure-filtered through
~ 0.4-um Nuclepore filter
4. Batch 4: Citrate
buffer
0.5 M citrate buffer
Batch: rotary
extractor
None
5. Up-flow column Deionized distilled water** Column, upward flow None
Pressure-filtered through
0.4-um Nuclepore filter
Leachate from column directly
passed through XAD-2 resin
* Factors such as particle size (<9.5 mm), extraction at room temperature, extraction time (24 h for batch extractions or until an effective 1:20
sol id: liquid contact ratio is reached for column extraction), one extraction on each waste, and effective 1:20 solid:!iquid ratio remained constant
**ASTM, Type I Reagent Water
buffer and to a conductivity of 20 mS/cm
using sodium chloride. The adjusted
extract (500 ml_) was then passed
through a gravity feed cartridge
containing XAD-2 resin (4.2 mL) which
was obtained in pre-cleaned, pre-filled
form from Isolab, Inc. (Akron, OH). The
column extracts were isolated directly on
16.8-mL XAD-2 resin cartridges. The
organics were eluted from the XAD-2
resin cartridge using acetone and methyl-
ene chloride. The resulting concentrates
were analyzed by GC and GC/MS using
fused silica capillary columns.
To assess the capabilities of the
procedures to remove organic
compounds, chromatograms from the
extracts were compared in two ways.
First, the total area of the chromatogram,
excluding the solvent peak (termed total
chromatographable organics or TCO),
was compared as a measure of the
relative mass extracted by the extraction
procedures. Second, the quantities of
individual compounds identified by the
chromatograms of the extracts were
compared. While it was not possible to
obtain absolute quantitative data on the
individual compounds, the equivalent
treatment of all extracts ensured a
reliable comparison. Blanks were run on
all procedures by carrying the media
through all extraction and analytical
operations. Maximum contaminant
(blank) levels below 10 ppb were
routinely obtained.
Task 2
The objective of Task 2 was to compare
two techniques for isolating organic
mutagens from sol id waste leachates and
wastewaters for testing in the Ames
Salmonella mutagenicity assay (Epler et
al. 1980). The two recovery techniques
that were compared were a resin
adsorption technique using Amberlite
XAD-2 resin and a solvent partition
technique using methylene chloride. The
XAD-2 resin technique (also used in Task
1) has been used extensively at the Oak
Ridge National Laboratory (ORNL) (Epler
et al. 1980) and at EPA's Health Effects
Research Laboratory at Research
Triangle Park, NC, while the solvent parti-
tion scheme is the one developed by the
EPA'S National Enforcement Investiga-
tions Center (NEIC), Denver, CO (EPA
1980b). Two known mutagens were used
as markers representing the basic (9-
amino acridine) and neutral
[benzo(a)pyrene] chemical classes. The
mutagens were added to four different
aqueous media (distilled water, a solid
waste EP extract, a "real-world" landfill
leachate, and an industrial wastewater)
to give a large variety in terms of physical
characteristics and possible chemical
interferences. The objective of this task
was to determine if significant differences
result in analytical recovery as well as in
the Ames Salmonella mutagenicity assay
when using either of the two preparation
protocols.
Task 3
The comparability of two laboratory
extractors (magnetically stirred and
rotary) used to stir and agitate solid waste
suspensions were examined for Task 3.
The magnetically stirred extractor at a
high and a low mixing rate and the rotary
extractor at 29 rpm were compared for
conducting the EP. The low mixing rate
used in the magnetic stirrer was the
lowest speed that could be achieved
while still keeping solids suspended, and
the high mixing rate was the highest
speed at which the stir bar could be
controlled.
A factorial experiment was conducted
using the three mixing rates and two
wastes, utilizing four extraction
replicates and three analytical
determinations of each extract. Two
statistical treatments of the data were
performed: variance component analysis
and Duncan's multiple range test.
Task 4
The objective of this task was to
evaluate the analytical constraints and
interpretations associated with using the
proposed reverse-phase High-Pressure
Liquid Chromatography (HPLC) protocol
(USEPA 1978) to assess the bioaccumu-
lation potential of solid waste extracts.
For many organic compounds, there is a
positive correlation between their
octanol/water partition coefficient and
their bioaccumulation potential (i.e.,
compounds with high octanol/water
partition coefficients have the potential to
accumulate biologically). A linear
relationship exists between octanol/
water partition coefficients and the log of
reverse-phase HPLC retention times.
Thus, plotting the log of reverse-phase
HPLC retention times of organic concen-
trates from solid waste extracts (from
Task 1) against the log of the
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octanol/water partition coefficients of
known compounds should provide a
method to estimate the potential for the
solid waste extracts to accumulate
biologically.
Results
Task 1
The relative effectiveness of the five
extraction procedures to extract TCO and
individual organic compounds from the
wastes is presented in the full report. The
column extraction procedure proved to be
more effective than any of the other
extraction procedures examined. In terms
of TCO in the extracts, the column extract
contained, on the average, greater than
10 times more organic material than
extracts from the EP.
To evaluate the relative effectiveness
of the extraction procedures to extract
organic compounds (using TCO and
individual organic compound values),
each procedure was ranked from lowest
to highest (1 to 5) for each waste. This
ranking scheme was used to statistically
detect significant differences (P < 0.05)
among extraction procedures across
wastes using a completely randomized
design. Applying this ranking scheme
revealed significant differences among
procedures, as shown in Table 2. The
following conclusions can be made
relative to the effectiveness of the five
extraction procedures to remove organic
compounds: (a) the column procedure
extracted more organic material than any
of the batch procedures, and (b) among
the batch extraction procedures,
deiomzed distilled water was the most
aggressive medium.
The major differences in quantities of
organic compounds extracted were
attributed to differences in leaching
procedures rather than leaching media
(except distilled water in batch
procedures which yielded higher
quantities of organics than other leaching
media). For example, the most important
features of the extraction procedures
were the vessels (column vs open and
closed containers) and techniques used
to separate the extract from the solid
portion of the waste (e.g., filtering
medium and pore size).
The five aqueous extraction procedure
extracts were found to be compatible with
the Sa/mone//a/microsome assay. More
of the wastes were determined to be
mutagenic from extracts obtained with
the Na-resin procedure than with any of
the other four aqueous extraction
procedures. Samples containing higher
levels of organic compounds (i.e., column
Table 2. Relative Flanking of the Extraction
Procedures for TCO and Individual
Organic Compound Values
Extraction Procedure
Mean Rank
Score
Total chromatographable organics
Column extraction
Batch extractions
Water
EP
Citrate buffer
Na-resin
5.00s
3.33h
2.33C
2.33°
2.00°
Individual organic compounds
Column extraction
Batch extractions
Water
Citrate buffer
Na-resin
EP
4.87*
3.13"
2.49t>'c
2.44
2.08'
b.c
a,b,c Mean rank scores with common super-
scripts are not significantly different (P < 0.05)
from one another by Duncan's multiple range
test.
extracts) exhibited non-specific cytotox-
icity which masked mutagenic activity in
these samples.
Task 2
The results of the recovery study for
isolation of organic compounds in Task 2
showed the extraction efficiency of the
resin technique was, in general, less
dependent on the aqueous media than
was solvent partition. Statistically signifi-
cant differences in the recovery of the two
mutagens from the aqueous media tested
were noted using solvent partition. By
contrast, no significant differences in the
recovery of the mutagens from any aque-
ous medium were noted using resin
adsorption. The data suggest that the
XAD-2 resin adsorption technique is
preferable to solvent partition considering
the wide variety of aqueous media en-
countered in hazardous waste testing/
monitoring. This would be a likely situa-
tion in the case of solid waste leachates in
that the chemical composition of the
leachate is highly dependent on the phys-
icochemical characteristics of the waste.
The mutagenic activity was directly
related to the chemical distribution of
benzo(a)pyrene and 9-amino-acridine.
Other organic species in the extracts did
not interfere with mutagenic testing in
either of the isolation techniques.
Qualitatively, the mutagenic activity of
the spiked extracts was identical to the
results obtained with positive control
samples of the mutagens. The resin
isolation procedure was also considered
superior from a laboratory performance
standpoint (i.e., time, application to multi-
ple samples, and cost of reagents).
Task 3
Extracts produced by the magnetically
stirred extractor (open container) and
rotary extractor (closed container) from
two solid wastes used in Task 3 showed
significantly different (P < 0.05) concen-
trations of Cd, Fe, Ni, As, and Zn. Neither
method yielded consistently high concen-
trations. However, because the concen-
trations of elements in these samples
were very low (ppb range), additional
samples containing higher levels of
extractable constituents should be
studied. If volatile constituents are of
interest, the closed rotary extractor may
be the preferred method to avoid losses
that may occur with an open container.
The closed system does not, however,
allow automatic pH adjustment.
Task 4
The extracts produced from the five
extraction procedures on nine wastes
(from Task 1) were tested for potentially
bioaccumulative materials in Task 4. Of
these extraction procedures, only the
column procedure produced extracts that
were considered potentially bioaccumu-
lative according to the proposed HPLC
protocol. The bioaccumulation potential
test itself was found to have several
drawbacks. No UV detector is universal
(therefore, non-absorbing materials may
be missed, e.g., chlorinated aliphatic
hydrocarbons), nor is the response of the
UV detector uniform (highly absorbing
materials may be given an overly high
ranking). Therefore, information obtained
is essentially qualitative. However, for
the present purposes, the test does
provide a screening method for the
detection of potentially bioaccumulative
organic compounds.
Summary
The following conclusions are based on
major results obtained for this research.
Task 1
• Ranking the five extraction proce-
dures from lowest to highest (1 to 5)
for ability to extract total chromato-
graphable organics (TCO), the
column procedure produced
extracts higher in TCO than batch
extracts (statistically significant
P < 0.05). Among the four batch
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procedures distilled water
extracted the most TCO. No signifi-
cant differences were observed
among the EP, Na-resin, and citrate
buffer extractions.
Task 2
• XAD-2 resin adsorption is superior
to solvent partition for recovery of
mutagens fom solid waste
leachates and wastewaters, due to
decreased dependence on the
chemical composition of the
aqueous matrix.
• XAD-2 resin adsorption has addi-
tional advantages of speed and
economics, particularly with large
numbers of samples.
Task3
• The magnetic stirrer agitation
method produced statistically
different inorganic concentrations
in EP extracts than those produced
by the rotary extractor. Neither
method showed consistently
higher concentrations.
Task 4
• The bioaccumulation potential test
has limitations in the analysis of
solid waste leachates. No quanti-
tative data can be generated with-
out prior knowledge of sample
components, and non-UV-absorbing
bioaccumulative materials such as
chlorinated aliphatic hydrocarbons
are not detected.
References
Epler, J. L, F. W. Larimer, T. K. Rao, E. M.
Burnett, W. H. Griest, M. R. Guerin, M.
P. Maskarinec, D. A. Brown, N. T.
Edwards, C. W. Gehrs, R. E. Millemann,
B. R. Parkhurst, B. M. Ross-Todd, D. S.
Shriner, and H. W. Wilson, Jr. 1980.
Toxicity of Leachates. EPA-600/2-80-
057, U.S. Environmental Protection
Agency, Washington, DC. 134 pp.
U.S. Environmental Protection Agency.
1978. Hazardous waste: Proposed
guidelines and regulations and
proposal on identification arid listing.
43 Fed. Regist. 58966.
U.S. Environmental Protection Agency.
1979. National interim primary
drinking water regulations. 40CFR 141.
U.S. Environmental Protection Agency.
1980a. Identification and listing of
hazardous waste. In: Environmental
Protection Agency Hazardous Waste
Management System. 40 CFR 261.24.
U.S. Environmental Protection Agency.
1980b. Microbial bioassay for toxic and
hazardous material. EPA-330/9-80-
002, Denver, Colorado.
D. K. Brown. M. P. Maskarinec. F. W. Larimer, and C. W. Francis are with
Oak Ridge National Laboratory, Oak Ridge, TN 37830.
Llewellyn R. Williams is the EPA Project Officer (see below).
The complete report, entitled "Mobility of Organic Compounds from
Hazardous Wastes," (Order No. PB83-163 956; Cost: $ 19.00, subject to
change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Environmental Monitoring Systems Laboratory
U.S. Environmental Protection Agency
P.O.Box 15027
Las Vegas, NV 89114
•fr U. S. GOVERNMENT PRINTING OFFICE: 1983/659-095/1915
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