"'
United States
Environmental Protection
Agency
Robert S. Kerr Environmental
Research Laboratory
Ada OK 74820
Research and Development
EPA-600/S2-84-135 Sept. 1984
Project Summary
Use of Short-Term Bioassays to
Evaluate Environmental
Impact of Land Treatment of
Hazardous Industrial Waste
K. W. Brown, K. C. Donnelly, and J. C. Thomas
A four-phase study was conducted to
evaluate the utility of short-term bio-
assays in monitoring the environmental
impact of land treatment of hazardous
industrial waste. During phase one,
bioassays were conducted using Bacil-
lus subtilis. Salmonella typh/murium.
and haploid and diploid forms of Asper-
gillus nidulans to define the chronic
toxic potential of each waste selected
for study. The acid, base, and neutral
fractions of each of the three wastes
studied induced genetic damage in at
least two of the three bioassays.
Phase two involved adding 2-nitro-
fluorene or benzo(a)pyrene to the soil.
This phase was conducted to evaluate
efficiencies of the blender and Soxhlet
extraction procedures, as well as poten-
tial interactions between known muta-
gens and soil components. Results
indicate that while greater quantities of
hydrocarbons were extracted using the
Soxhlet method, there was no appreci-
able difference in mutagenicity of the
extract using either procedure. In addi-
ion, when pure compounds were added
to the soil, the extraction efficiency
using the blender procedure averaged
greater than 85%. as measured by High
Pressure Liquid Chromatography
(HPLC). There was no statistical dif-
ference in mutagenicity of the pure
compound or the extract of the soil plus
the compound.
Phase three consisted of a greenhouse
study in which each of three wastes was
applied to two soils. Soil and runoff
samples were collected at various times
over a 360-day or a 840-day interval.
Results from chemical analyses indi-
cated that waste constituents were
degraded in the soil. Bioassays of soil
and water extracts indicated increased
mutagenic activity, caused perhaps by
the degradative process forming direct-
acting mutagens and converting indi-
rect-acting mutagens to direct-acting
compounds. When compared on an
equivalent volume basis, the mutagenic
potential of waste-amended soils was
reduced over time, and in some cases, it
was reduced to a nonmutagenic level.
A wood-preserving bottom sediment
was applied to barrel-sized lysimeters in
the final phase of the project to compare
results of soil-core and soil-pore liquid
monitoring. Leachate and soil samples
were collected prior to as well as 30 and
90 days after waste application. Differ-
ent types of compounds were detected
in soil-core and soil-pore liquid samples.
These results indicate that if a land
treatment facility is not properly man-
aged, mutagenic constituents from
land-applied waste may migrate through
the soil.
This study was undertaken to demon-
strate that short-term bioassays can be
used to trace the environmental fate of
mutagenic constituents in land applied
hazardous industrial wastes.
This Project Summary was developed
by EPA's Robert S. Kerr Environmental
Research Laboratory, Ada, OK, to an-
nounce key findings of the research
project that is fully documented in a
separate report of the same title (see
Project Report ordering information at
back).
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Introduction
Over the past three decades, concurrent
with the development of numerous new
chemicals, the United States has experi-
enced rapid industrial expansion, with its
inevitable byproduct, generation of large
volumes of hazardous wastes—150 mil-
lion metric tons annually according to a
1983 estimate by the U.S. Environmental
Protection Agency (EPA).
Recently, in many areas, the three most
widely used disposal methods—deep sea
dumping, incineration, and landfilling—
have been replaced by land treatment,
i.e., incorporation of waste into the sur-
face layer of soil resulting in the degrada-
tion or attenuation of hazardous waste
constituents.
Final interim regulations promulgated
by the EPA (1982) state that a waste
cannot be land applied unless the waste
is rendered less or nonhazardous by
chemical or biological reactions in the
soil. Before land treatment can become a
viable method of hazardous waste man-
agement, techniques are needed for the
monitoring of hazardous constituents and
their metabolites. Use of a combined
biological and chemical testing protocol
may provide the most practical means of
efficiently monitoring a hazardous waste
land treatment site. Use of chemical
analyses alone fails to account for inter-
actions of components of a complex
mixture, production of mutagenic metab-
olites via degradative pathways, and
chemical reactions between nontoxic
precursors that may result in formation of
mutagenic compounds. An appropriately
selected bioassay should be capable of
integrating these effects. Use of biological
analyses alone, however, could fail to
account for artifacts generated in the
collection or extraction process.
The objectives of this current research
were to characterize genotoxic constitu-
ents of three hazardous wastes, monitor
waste degradation in soil, and determine
the environmental fate of mutagenic
waste constituents following land appli-
cation. The project was divided into four
main phases to meet these objectives,
and to develop a set of test protocols to be
used to monitor environmental contam-
ination. The first phase, waste charac-
terization, included an acute toxicity
evaluation of ten wastes, a complete
characterization of the mutagenic poten-
tial of seven subf ractions of three selected
wastes, and a chemical characterization
of major organic constituents. In phase
two, direct- and indirect-acting mutagens
were added to the soil in order to quantify
extraction procedures and determine the
effect of soil components on the activity of
mutagenic compounds. Phase three con-
sisted of a greenhouse study in which
three wastes were applied at one loading
rate to two soil types packed in boxes.
Simulated rainfall was applied, and runoff
and soil samples were collected at various
time intervals during a 360- or 540-day
experimental period.
Results from phase three of the project
were used to evaluate the effect of
degradation on the mutagenic activity of
waste-amended soil and the potential for
removal of mutagens in runoff water. In
the final study phase, one waste was
applied at one loading rate to an undis-
turbed soil enclosed in lysimeters. Move-
ment of mutagens through soil was
monitored by collecting soil-core and soil-
pore liquid samples.
Materials and Methods
Wastes
Initially, thirteen wastes were collected
for use in the project. Included were two
wood-preserving wastes, four refinery
wastes, four petrochemical wastes, a pulp
and paper waste, an alum sludge, and a
paint sludge (Table 1). Selection of three
wastes for use in the waste characteriza-
tion and greenhouse studies was based
on results from chemical characterization
and acute toxicity testing.
Extract/on
Two methods were used for extraction
of hydrocarbons from wastes and from
waste-amended soils. The majority of
samples were extracted using the blender
technique. Comparisons were made with
a limited number of samples using a
Soxhlet extractor.
Dichloromethane was selected from a
group of agents to extract organic frac-
tions of the wastes and soil. Dichloro-
methane consistently provided the great-
est extraction efficiency for the type of
anticipated materials. Hydrocarbons were
extracted from the waste or waste-
amended soils using procedures described
in the literature and partitioned into acid,
base, and neutral fractions.
Waste Extraction
Runoff samples were extracted and
passed through a mixed bed of 4.0 g of
XAD-2 and 6.3 g of XAD-7, or approxi-
mately 20 cm3 of each resin. After loading
the water sample, dry nitrogen was
introduced into the column to remove the
residual aqueous phase.Thecolumnwas
washed with 120 ml of distilled water to
remove residual histidine. The adsorbed
organic compounds were eluted with 160
ml of acetone.
Chemical Analysis
Chemical analyses of waste and soil-
waste extracts were conducted by the
Table 1. Gross Characteristics of Hazardous Wastes Collected for Study
Waste
Wood-Preserving Bottom
Sediment (PENT S)
Wood-Preserving Wastewater
Slop-Oil Emulsion So/ids
Combined API Separator, Waste
Treatment Sludge (COMBO)
Storm-Water Runoff
Impoundment (SWRI)
Dissolved Air Flotation
Acetonitrile
Methyl Ethyl Ketone
Phenol Production
Agricultural Chemicals —
Biosolids Waste
Primary Clarifier Pump and
Papermill
Alum Sludge
Paint Sludge
Extractable'1
Hydrocarbons
EPA No. (%)
K001 27
NT
K049 86
K051 41
21
K048 5
K013 2
97
0.2
0.2
NT
NT
NT
Physical 2
Form
Sludge
Liquid
Liquid
Sludge
Sludge
Sludge
Liquid
Liquid
Liquid
Liquid
Solid
Liquid
Sludge
Use in
Study3
A.W.G.L
A
A.W
A.W.G
A.W.G
A
A.W
A.W
A
A
N
N
N
1* Percent by weight, extracted with dichloromethane; NT = not tested.
'Physical form estimated from visual observation.
3A = acute toxicity; W = waste characterization; G = greenhouse; L = lysimeter; N = not used.
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EPA's Robert S. Kerr Environmental
Research Laboratory. Compounds were
identified using a Finnigan OWA Auto-
mated GC/MS. The GC capillary column
used was a J & W Scientific DB-5-30W.
Biological Analysis
The ability of samples to induce genetic
damage was measured in three microbial
test systems. A eukaryotic bioassay em-
ploying Aspergillus nidulans (a fungus)
was used to detect point mutations and
small deletions induced in a haploid
genome. The diploid organism was used
to detect chromosome aberrations, mitotic
recombination, gene mutation, nondis-
junction recombinogenic events, reces-
sive lethals, and spindle poisons. A
sample was considered mutagenic if there
was a positive slope on the mutation
induction curve, or if the induced mutation
frequency for at least two exposure times
was more than twice the spontaneous
mutation frequency.
A microbial DMA repair assay was used
to measure the capacity of a sample to
produce increased lethal damage in DNA
repair deficient strains. Six strains of B
subtilis deficient in different recombina-
tion (Rec~) and/or excision (Exc~) repair
were used to test for lethal DNA damage.
These included Rec'stramsrec,45, recE4;
mc-1, Exc" strain hcr-9; and Rec'/Exc"
fh2006.7. All of these strains are isogenic
with B subtilis strain 168 which has all
repair intact A response was considered
positive if the distance of growth inhibi-
tion was more than 2 5 mm greater in one
of the repair deficient strains than in the
repair proficient strain 168. Fractional
survival (N/No) was determined for those
strains showing the greatest sensitivity
(inhibition) to the test chemical
A Salmonella/microsome assay was
used to evaluate mutagenic activity of
waste fraction samples
Results and Discussion
Biological and chemical analyses were
employed in the study to evaluate muta-
genic potential of the acid, base, and
neutral fractions of three hazardous
industrial wastes A summary of the
results obtained in the different biological
test systems is provided in Table 2
For the wood-preserving bottom sedi-
ment, the maximum level of genotoxic
activity was detected in the base fraction
With metabolic activation, the base frac-
tion induced the maximum response in
the B subtilis DNA repair assay, the
Salmonella/microsome assay (strains
TA98, TA100, TA1538), and the Asper-
Table 2. Summary of Results Obtained from Testing Waste Fractions in Biological Test
Systems
Bioassay1*
Sample
PENTS
SWRI
COMBO
DNA SALM
S93 - + _ H
Acid + + - •>
flase _ ++ -M
Neutral i
Acid - - . +^
Base *
Neutral +•»
/4c/d + - +•»
Base - ' - - -H
Neutral - - + +-»
BACPM ASPMT ASPDP
>
_
•
•
0
0
0
0
0
0
+ - H
+ + +K
+ + +H
- + H
0 + +^
0 + +^
0 + H
0 + H
0 + H
0 + +^
'
+ 0
± 0
i- + 0
^ + 0
^ + 0
+ 0
+ 0
± 0
H + 0
- B. subtihsD/Vxl repair assay; SALM = S. typhimunum reverse mutation assay; BACPM = B.
subtillis reverse mutation assay; ASPMT = A nidulans methionine assay; ASPDP = A. nidulans
dip/oid assay
'Response 0 - not tested; - - <2 times background, ± =>2 <2 5 times background; + - >2.5 <5
times background, ++ =>5 times background
3S9 = 9000 x g supernatant from Aroc/or 1254 induced rats
gillus methionine assay. In the absence of
metabolic activation, the acid fraction
induced the maximum response in the
Aspergillus diploid assay and the Bacillus
DNA repair spot test. Chemical analyses
identified a variety of potentially genotoxic
waste constituents, including pentachlo-
rophenol. Thus, biological analyses de-
tected genotoxic compounds in the wood-
preserving waste, and chemical analyses
detected compounds that could be mobile
and recalcitrant. These results indicate
that land application of a wood-preserving
waste should proceed with caution, pre-
ferably at a low application rate.
Although chemical analyses were un-
able to identify any of the major organic
constituents of the storm-water impound-
ment (SWRI) waste, biological analyses
detected mutagenic activity in all three
waste fractions. The maximum response
observed m \heSalmonella andAspergil-
lis mutagenicity assays was' induced by
the acid fraction, while no response was
observed in the Bacillus DNA repair assay
with any of the fractions All three
fractions induced a positive response in
the diploid assay, with the maximum
response induced by the base fraction.
Thus, biological analyses detected geno-
toxic compounds in all three fractions of
the SWRI waste; however, these com-
pounds were present in quantities that
were below the detect ion limits of chemi-
cal analyses.
In the combined API separator/slop-oil
emulsion (COMBO) waste, results of
chemical and biological analyses indicate
that the neutral fraction had the greatest
mutagenic potential. Both mutagenesis
assays detected the maximum response
in the neutral fraction. The maximum
response in the DNA repair assay was
induced by the acid fraction, and the base
fraction induced the maximum response
in the diploid assay. These results indicate
that the COMBO waste contained com-
pounds capable of inducing a range of
genetic damage. Although chemical anal-
yses were unable to identify all of the
genotoxic waste constituents, both bio-
logical and chemical analyses indicated
that the neutral fraction has the greatest
mutagenic potential.
Biological analyses of the organic ex-
tract of three soils which have been used
solely for agricultural purposes demon-
strated the presence of low concentra-
tions of mutagens and potential carcino-
gens. Chemical analyses of two of the
three soils used m this research were
unable to conclusively identify the muta-
genic contaminants. However, the past
history of these soils indicates that the
most probable source of mutagenic activ-
ity is trace quantities of partially oxidized
residues from previous biocide applica-
tions. Mutagenic activity of these trace
contaminants also may have been en-
hanced by the presence of promoters and
cocarcinogens.
Biological and chemical analyses of
two soils amended with either 2-nitro-
fluorene or benzo(a)pyrene indicated that
the blender extraction procedure provides
adequate recovery of mutagenic com-
pounds. Results from the Salmonella/
microsome assay indicate that there was
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no appreciable difference in mutagenic
activity of the pure compound or extract of
the soil amended with the pure compound
at equivalent dose levels. In addition,
extraction efficiency as measured using
HPLC analyses averaged greater than
85% for both chemicals at all treatment
levels on both soils. This combined
chemical/biological analytical approach
has demonstrated that for the compounds,
levels and soils evaluated, there are no
interactions with soil compounds, and
that the blender procedure provides effi-
cient extraction of mutagenic compounds
from soil.
Degradation, infiltration, and removal
will influence the quality and quantity of
hazardous organic compounds in runoff
water from a land-treatment facility. In
order to evaluate influence of these
factors on mutagenic potential of runoff
water, results from waste-amended and
control soils were compared on the basis
of equivalent volumes. A comparison of
results from testing the equivalent of 10
ml of runoff water from the wood-
preserving waste-amended Norwood soil
in the Salmonella assay indicates that
mutagenic potential increases consistent-
ly from immediately after waste applica-
tion through 540 days after waste
application (Figure 1). Contrasting results
were observed in the Aspergillus assay
(Figure 2). In the wood-preserving waste-
amended Norwood soil, mutagenic activ-
ity of runoff water collected 360 days
after application decreased to a level
approximately 16% of that detected
immediately following waste application
(Figure 2). The surviving fraction in Asper-
gillus increased significantly over the
360-day period. If this effect also occurred
in the Salmonella assay, the induced
mutants per survivor measured immedi-
ately after waste application would be
three to four times greater than the net
reversion frequency, while the induced
mutants per survivor and the net mutation
frequency measured 540 days after appli-
cation would be approximately the same.
Thus, the results from the two bioassays
may be comparable.
In the Bastrop soil, the mutagenic
response in both bioassays decreased
from immediately after waste application
through 540 days after waste application
(Figures 2 and 3). In both bioassays, the
response induced by the sample collected
360 days after application was at a level
that was only slightly greater than twice
background. However, 540 days after
application, there was an appreciable
increase in both the amount of extractable
hydrocarbon and mutagenic potential of
2 60
NORWOOD PENTS
Runoff Water
C—Unamended
Hydrocarbor
fc
| 20"
IB
1
1 his* Revertants
o *w O)
5 O O
a
K.
c
C 7
I
P
L_
/
'OX
0
\ +
1
D
%//•
p
c
r— •'-
S9
S9
C 7
i 1777\
16
1
'OX
0
D
\
'///,
I
C
1
C
3<
/=
^
SO
>
!
-^/
i
5<
1
P
i —
10
D
^
1
Time (Days)
Figure /. Extractable hydrocarbons (mg/'I) and'mutagenic activity (revertants/'10 mlj in runoff
water from PENT S waste-amended soil.
runoff water from the wood-preserving
waste-amended Bastrop soil. The in-
creased mutagenicity observed in the
sample collected 540 days after applica-
tion may have been a result of degradation
increasing the reactivity of residual
compounds. Degradation may have re-
duced the concentration of nonmutagenic
compounds, while increasing the relative
concentration of mutagenic compounds.
The results obtained from the runoff water
from either soil amended with the wood-
preserving waste as measured with the
Salmonella and Aspergillus assays indi-
cate that significant levels of mutagenic
activity are detectable 360 days after
waste application. However, the overall
results indicate that the mutagenic poten-
tial of the runoff water was appreciably
reduced 360 days after waste application.
In order to determine if a waste is
rendered less or nonhazardous by soil
incorporation, a comparison of mutagenic
potential of equal volumes of waste-
amended soil is necessary. In the Salmo-
nella assay, the mutagenic potential
(Figures 4 and 5) was determined by
calculating the mutagenic activity ratio of
two nontoxic dose levels from a five
member dose-response curve and adjust-
ing according to the rate of degradation.
Utility of these data lies in their ability to
define hazardous characteristics of a
waste-amended soil. By comparing mu-
tagenic potential of equivalent volumes of
waste-amended soil over time, determi-
nation of whether a waste is rendered
less or nonhazardous by soil incorporation
is possible.
Results from evaluating the effect of
soil degradation on the mutagenic poten-
tial of wood-preserving waste-amended
soils are presented in Figures 4 and 5. In
the Norwood soil, mutagenic potential of
the base and neutral fractions decreased
to below the significant level in both
bioassays. In the acid fraction, mutagenic
potential with metabolic activation was
reduced to below a level at which the
response would be considered mutagenic
in both bioassays. In contrast, the re-
sponse without activation in Aspergillus
was increased by more than 50% from
immediately after waste application to
360 days after application. Mutagenic
potential of the neutral fraction from
wood-preserving waste-amended Bastrop
soil was also decreased to below signifi-
cant levels in both bioassays. In the acid
and base fractions from wood-preserving
waste-amended Bastrop soil, mutagenic
potential increased during the 180 days
following waste application. The response
in Aspergillus from both fractions was
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40-1
o 20 -
§
+S9
-S9
40-1
CONTROL.
10 ml
PENTS.
2.50 ml
20-
NW
BA
Not Tested
ISO
NW
360
Time IDaysl
Time (Days)
Figure 2.
Total mutation frequency per 10(
waste-amended soils
survivors in A nidulans induced by the extractable hydrocarbons in runoff water from PENTA S
c
o
-Q
-S;
-Q
-------
80-
40
10
5
NW
N
B
A
BA
N
B
A
NW
N
B
BA
N
B
NW
N
P
A
B
N
BA
N
A
ENTS (EH)
— Acid
—Base
—Neutn
NW
N
il
BA
N
20-
5-
+S9 ffl
-S9 a
NW
V77* NW BA
*VV/ wJJt rVVvt ryvVi
o v% O~M pW M ^1W
B
A
'%
1
yO'V
^
R2
iny
B
A
I
/^x2
r^
20 -,
«t 10-
5
BASE, WOmg
20
^ 10
NW BA
BA NW BA
BA
-180 4 b 360-
Time (Days)
NEUTRAL 10 mg
Time (Days)
Figure 4. Total extractable hydrocarbons and mutagenic potential of equivalent volumes of RENTS waste-amended Norwood (NW) andBastrop
(BA) soils as measured with S. typhimurium strain TA98 with and without metabolic activation. Dashed line (—) is equal to 2.5 times
solvent control.
consistently identified in the waste and
soil-core samples at all depths.
When compared to soil-core samples,
analyses of soil-pore liquid samples pro-
vides a slightly different perspective on
the capacity of wood-preserving waste
samples to migrate through soil. Biolog-
ical analysis with S. typhimurium strain
TA98 indicated no detectable mutagenic
activity in the leachate water collected
from control and waste-amended lysim-
eters prior to waste application. Mutagen-
ic potential of the soil-pore sample
collected at a depth of 90 cm 30 days after
application was approximately seven
times greater than the control. The sample
collected 90 days after application had a
mutagenic potential approximately ten
times the control (Figure 7). Thus, the
bioassays detected significantly greater
quantities of mutagenic activity in soil-
pore samples from wood-preserving
waste-amended lysimeters than in sam-
ples from control lysimeters, both on 30
and 90 days after waste application.
There were ten compounds in soil-pore
samples from wood-preserving waste-
amended lysimeters, including anthra-
cene and pentachlorophenol which were
also detected in the 0 to 15 cm soil-core
sample collected 90 days after waste
application. Tetrachlorophenol was also
detected in the leachate sample collected
90 days after application. This compound
was not present in waste- or soil-pore
samples. As a result, tetrachlorophenol
may have been transformed from penta-
chlorophenol at the soil-water interface
and subsequently leached into the soil-
pore water 90 cm below the soil surface.
Analysis of samples from lysimeters
amended with wood-preserving bottom
sediment indicated that mutagens and
potential carcinogens are capable of
migrating to a depth of 75 cm below the
zone of incorporation within 90 days
following waste application. Mobility of
waste constituents may have been influ-
enced by the high waste application rate
and high amount of rainfall (12.66 cm)
that occurred during the 90-day study.
Conclusions
This research has demonstrated the
utility of a combined testing protocol using
biological analyses to measure genotoxic
potential of waste fractions and chemical
analyses to identify major organic consti-
tuents. These results have also demon-
strated the inability of chemical analyses
to provide a comprehensive evaluation of
the genotoxic potential of a hazardous
industrial waste. While it is possible that
more intensive chemical analyses could
have identified genotoxic compounds
present in trace concentrations, informa-
tion would still be lacking as to inter-
actions of waste constituents. However,
the results also indicate that chemical
analyses are a necessary component of a
hazardous-waste analytical protocol.
Chemical analyses are necessary to iden-
tify waste constituents and to verify the
absence of artifacts generated in the
collection or extraction process.
The efficiency of the blender technique
for extracting the diagnostic mutagens,
2-nitrofluorene or benzo(a)pyrene, aver-
aged greater than 85% as measured by
HPLC. In addition, there was no appreci-
able difference in the mutagenic activity
of the pure compound and the extract of
soil amended with the pure compound.
Results from the greenhouse study
indicate that mutagenic potential of runoff
water from hazardous waste-amended
soils should eventually return to back-
ground levels. Major factors influencing
mutagenic potential of residual hydro-
carbons at a land treatment facility include
the number of different compounds pre-
sent, concentrations of these compounds,
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PENTS
+S9 {S3
-S9 CH
ACID (200 mg)
Time (Days)
Figure 5. Total induced mutation frequency of equivalent volumes of PENT S amended
Norwood (NW) and Bastrop fBA) soils as measured in A. nidulans methionine
system with and without metabolic activation. Dashed line I—) is equal to total
induced mutation frequency of 5.0/10* survivors.
This research has demonstrated that
land treatment can be a viable disposal
option for a variety of hazardous wastes.
However, there is also an indication that
mutagen and potential carcinogens can
be released in runoff or leachate water.
This information makes it imperative that
a treatment demonstration, as suggested
by the EPA in 1982, precede the applica-
tion of hazardous waste to soil. This
treatment demonstration should include
degradation and leaching studies, as well
as a demonstration of volatilization rates
and the effects of management practices
and repeat applications. Bioassays may
be used a an integral part of the treatment
demonstration and, more importantly, in
operational monitoring of an active site.
and toxic effects and interactions of these
compounds. Results indicate that differ-
ent soils will have substantially different
capacities to retain and degrade organic
compounds. Therefore, bioassays provide
an effective analytical tool for evaluating
when mutagenic potential of runoff water
or soil from a hazardous waste land
treatment facility has returned to back-
ground levels. While these results can
only be applied to the wastes, soils, and
loading rates employed, they do indicate
that land treatment can render a waste
less hazardous or nonhazardous through
degradation or transformation in the soil.
Recommendations
In order to obtain the most accurate
evaluation of hazardous characteristics
of a waste, both chemical and biological
analyses should be used. The best results
wou Id be obta i ned if a battery of bioassays
were used to define the genetic toxicity of
a hazardous industrial waste. Bioassays
used should include some that are capable
of detecting point mutations, DNA repair
damage, and chromosome damage. In
addition, the testing protocol used to
monitor hazardous-waste land treatment
should include both chronic and acute
toxicity bioassays.
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1200-
1000
I 6°
o
s
< 40
3
20
10,171
PS
I
1
1
0-15
Lysimeter Soil
\ \ Control—C E3
CH PENTS—PS ^
-S9 Day 90 -S9
PS
I
1
PS
15-45
Depth (cm)
45-90
\
Waste
PS
/u
!
1
ocarbons
§
actable Hydr
co
Cl
x
Uj
1.0
C
0-1i
Lysimeter Soil
Control— C
RENTS— PS
PS
C PS
C
15-45 45-90
Depth /cm)
Figure 6. Extractable hydrocarbons andmutagenic activity from soil-core samples collected at
various depths on day 90.
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600 n
ti-
ll)
^200
-C-i
Lysi meter Leach ate
Control C
PENTS PS
-S9
+S3
-PS-
I Y////A I V///A
-PS-i
I
-PS-
Background
Day 30
Day 90
Figure 7. Mutagenic activity of leachate water from control and PENT S waste-amended
lysimeters.
K. W. Brown, K. C. Donnelly, and J. C. Thomas are with Texas Agricultural
Experiment Station, Texas A&M University, College Station, TX 77843.
John E. Matthews is the EPA Project Officer (see below}.
The complete report, entitled "Use of Short-Term Bioassays to Evaluate
Environmental Impact of Land Treatment of Hazardous Industrial Waste,"
(Order No. PB 84-232 560; Cost: $29.50, 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:
Robert S. Kerr Environmental Research Laboratory
U.S. Environmental Protection Agency
Ada, OK 74820
*USGPO: 1984-759-102-10685
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