United States
Environmental Protection
Agency
Process Measurements Branch
Industrial Environmental Research
Laboratory
Research Triangle Park NC 27711
EPA-600/8-7 9-021
August 1979
vvEPA
Biological Screening of
Complex Samples From
Industrial/Energy Processes
Cooperative Project of IERL-RTP
and EPA Health and Ecological
Laboratories
-------
RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of
environmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the SPECIAL REPORTS series. This series is
reserved for reports which are intended to meet the technical information needs
of specifically targeted user groups. Reports in this series include Problem Orient-
ed Reports, Research Application Reports, and Executive Summary Documents.
Typical of these reports include state-of-the-art analyses, technology assess-
ments, reports on the results of major research and development efforts, design
manuals, and user manuals.
EPA REVIEW NOTICE
This report has been reviewed by the U.S. Environmental Protection Agency, and
approved for publication. Approval does not signify that the contents necessarily
reflect the views and policy of the Agency, nor does mention of trade names or
commercial products constitute endorsement or recommendation for use.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
-------
Biological Screening of
Complex Samples From
Industrial/Energy Processes
A Cooperative Project of IERL-RTP
and EPA Health and Ecological
Laboratories
Process Measurements Branch
Industrial Environmental Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC
June 1979
-------
BIOLOGICAL SCREENING OF COMPLEX SAMPLES
FROM INDUSTRIAL/ENERGY PROCESSES
INTRODUCTION
The earliest indicators of the dangerous nature of an unknown substance were
biological. Sudden fish kills often indicated point source contamination of water
supplies. In Japan, a plastics factory discharging wastes containing mercury into
Minimata Bay ultimately caused 46 human deaths and poisoned another 120 per-
sons. In a highly industrial society, it is essential that these hazardous discharges be
detected and controlled before they cause gross and sometimes irreversible damage
to the environment. The biological testing scheme incorporated as part of the
EPA/IERL-RTP's environmental assessment procedures is designed for this purpose.
The biological testing protocols discussed in this report are the result of a cooper-
ative effort by personnel from the health and ecological effects laboratories of the
Environmental Protection Agency working with personnel from IERL-RTP. The
cooperative programs were initiated in 1974. In 1976, a subcommittee represent-
ing EPA health and ecological laboratories was formed. Members of this subcommit-
tee, listed in Table 1, prepared the test protocols and evaluated results obtained on
complex process samples from three pilot studies. These studies were completed in
1978.
THE PHASED APPROACH TO ENVIRONMENTAL ASSESSMENTS
EPA's Industrial Environmental Research Laboratory at Research Triangle Park
(IERL-RTP) has developed an environmental assessment scheme for the detection of
hazardous materials in industrial effluents and gaseous emissions. lERL-RTP's en-
vironmental assessment scheme incorporates five major components: technology
background development; sampling and analysis; environmental goals; impact
analysis; and control technology evaluation. The sampling and analytical scheme
couples a battery of biological indicators with a series of chemical tests to
characterize the nature of a stream in terms of hazardous materials. This scheme in-
volves three levels of effort; Level 1 is a screening phase for the detection of any ef-
fluent or emission that might be detrimental to the environment; Level 2 involves
verification of the hazardous properties, indicated by Level 1 tests, and specific
identification of chemical components of the stream; and Level 3 is a long-term
monitoring phase, initiated to determine variations of the hazardous materials in an
industrial stream with time and to evaluate the efficiency of control technologies.
Note that an indication of possible hazard in Level 1 chemistry or biology dictates
proceeding to Level 2, where positive results are confirmed and false positives
screened out. Level 3 then monitors selected chemical and biological indicators over
a period of time. Figure 1 graphically presents the phased approach.
-------
TABLE 1. ENVIRONMENTAL ASSESSMENT STEERING COMMITTEE:
BIOASSAY SUBCOMMITTEE MEMBERS
David T. Tingey
Environmental Research Laboratory
Environmental Protection Agency
200 SW 35th Street
Corvallis, OR 97330
James A. Dorsey
Industrial Processes Division, MD-62
Industrial Environmental Research Laboratory
Environmental Protection Agency
Research Triangle Park, IMC 27711
William B. Horning
Newtown Fish Toxicology Station
3411 Church Street
Cincinnati, OH 45244
Joellen L. Huisingh
Health Effects Research Laboratory, MD-68
Environmental Protection Agency
Research Triangle Park, NC 27711
Raymond G. Merrill
Industrial Processes Division, MD-62
Industrial Environmental Research Laboratory
Environmental Protection Agency
Research Triangle Park, NC 27711
Gerald F. Stara
Health Effects Research Laboratory
Environmental Protection Agency
Cincinnati, OH 45268
Gerald E. Walsh
Environmental Research Laboratory
Environmental Protection Agency
Sabine Island
Gulf Breeze, FL 32561
Michael D. Waters
Health Effects Research Laboratory, MD-68
Environmental Protection Agency
Research Triangle Park, NC 27711
ShahbegSandhu
Health Effects Research Laboratory, MD-68
Environmental Protection Agency
Research Triangle Park, NC 27711
Michael A. Pereira
Health Effects Research Laboratory
Environmental Protection Agency
Cincinnati, OH 45268
-------
COMPLEX SOURCE SAMPLES
LEVEL
1
CHEMICAL
BIOLOGICAL
+
+
+
+
+
+
+
SCREENING ALL
STREAMS FOR
POTENTIAL
HAZARD
LEVEL
2
VERIFICATION AND
QUANTITATION
OF HAZARDOUS
COMPONENTS IN
INDICATED STREAMS
LEVEL
3
LONG-TERM
MONITORING
FOR VARIATION
IN OUTPUT AND/OR
EVALUATION OF NEW OR
ADDITIONAL CONTROL
TECHNOLOGY ON
INDICATED STREAMS
CHEMICAL
BIOLOGICAL
+
+
+
+
CHEMICAL
BIOLOGICAL
POSITIVE RESPONSE
INDICATES CONTINUED
CONCERN AND POTENTIAL
FOR HAZARD
Figure 1. The phased approach to environmental assessments.
-------
LEVEL 1 BIOLOGICAL TESTS
The Level 1 screening phase uses a series of short-term bioassays for the detec-
tion of acute biological effects (reference 1). The bioassays can be divided into
health-related tests and ecological tests. The health tests were selected to screen
for both acute toxic and potential chronic (i.e., carcinogenic) health effects. The ro-
dent acute toxicity test employs a quantal prescreen in rats followed by a quan-
titative assay. The cellular toxicity assay in mammalian cells measures cellular
viability or survival as well as more sensitive metabolic endpoints. The cells
employed in Level 1 are rabbit alveolar macrophage (RAM) for particulates, and
Chinese Hamster Ovarian (CHO) or WI-38 human embryonic lung cells for liquids.
Also included in the health tests is the Ames Salmonella typhimurium reverse muta-
tion assay. This assay uses bacteria to screen complex process samples for poten-
tially mutagenic or carcinogenic activity.
The aquatic ecological tests measure acute toxic effects of an industrial effluent
or emission on freshwater or marine fish (fathead or sheepshead minnows),
freshwater or marine invertebrates (Daphnia or grass shrimp), and selected species
of freshwater or marine algae. The aquatic ecological tests are "static" tests, in-
volving the exposure of the various organisms to several concentrations of an ef-
fluent sample in a closed system or aquarium. The organisms are monitored for ab-
normal responses or death.
Terrestrial ecological tests, including soil respiration, root elongation, plant stress
ethylene, and insect tests, are under evaluation; these tests are being developed and
will be part of the Level 1 protocol in the near future. The soil respiration test is bas-
ed on changes in normal uptake or output of carbon dioxide by soil microbes when a
solid or liquid toxic material is introduced into the soil. The root elongation test
evaluates samples via the inhibitory effect of toxic chemicals on seed germination
and root elongation. The stress ethylene test assays samples by way of the release
of ethylene by plants when the plants are exposed to toxic gases. The insect test
will measure the toxicity of solid, liquid, or gaseous samples on sensitive insect
species.
Although no single biological test can thoroughly predict the overall hazard-
ousness of a material, this battery of rapidly run biological tests on a wide variety of
biological receptors or indicators can indicate the kind of damage a material could
produce. Table 2 details the Level 1 biological tests. Figure 2 is a matrix of the ap-
plication of each Level 1 biotest to specific sample types.
The Level 2 biological tests are being developed to verify or confirm the Level 1
presumptive positive results and give more specific information on the nature of the
biological effect. As will be illustrated in the following section on case studies, the
Level 1 screening bioassays will prioritize streams for further testing so that the
available resources of time and funding can be directed toward the relatively few
hazardous streams. Where Level 1 chemical or biological tests indicate the need for
further analysis, Level 2 biological tests can be applied to more clearly define the
health or ecological hazard related to the stream in question. In some cases. Level 2
biological tests may be as simple as those in Level 1, while other cases will require
much more elaborate and classical methods for assessing biological hazard.
-------
TABLE 2. LEVEL 1 SCREENING BIOASSAYS
HEALTH EFFECTS TESTS
TEST
EFFECT
DESCRIPTION
TEST OUTPUTS
Microbial Mutagenesis
(Ames Test)
Mutagenesis
Genetically sensitive strains of microorganisms
are exposed to various doses of sample with and
without metabolic activation.
Mutagenic response is measured relative
to controls.
Cytotoxicity
Cellular Toxicity
Selected cells (RAM, CHO, orWI-38) are exposed
to various doses of sample, then various endpoints
are measured.
An index of functional impairment, toxic-
ity, and metabolic change is established
relative to controls.
Rodent Acute Toxicity
(RAT Test)
Whole Animal
Toxicity
Rats or other rodents are fed a quantity of sample,
then observed daily for adverse symptoms over a
14-day period. The experiment is terminated with
a necropsy exam.
Inventory of pharmacological and gross
physiological effects in a whole animal
system.
ECOLOGICAL EFFECTS TESTS
TEST
Algal Growth Response
EFFECT
Algal
Growth Inhibition
or Promotion
DESCRIPTION
Cultures of selected marine and/or freshwater algae
are used to gauge reaction to sample or dilution
thereof.
TEST OUTPUTS
Growth response measure stimulation
or inhibition.
Aquatic Animal Exposure
(Static Acute Bioassay)
Terrestrial
Animal Toxicity
Toxicity to
Plants, Insects, and
Soil Microbiota
Select marine and/or freshwater vertebrates and
invertebrates are exposed to a graded dilution series
of samples.
Tests in these three areas are under evaluation.
Gross index of toxic potential to representa-
tive animals.
Effects on plants, insects, and soil of complex
samples.
-------
SAMPLE FOR BIOLOGICAL ANALYSIS
oo
GASES AND SUSPENDED
PARTICULATE MATTER
GASEOUS
GRAB
SAMPLES
i
MICROBIAL
MUTAGENESIS,1
CYTOTOXICITY.1
STRESS ETHYLENE
1.2
PARTICULATES
h
i
Jim 1
N
1
ntnl
MICROBIAL
MUTAGENESIS,
RODENT ACUTE
TOXICITY,
CYTOTOXICITY,
TERRESTRIAL
BIOASSAYS1'2
SORBENT
EXTRACT
1
AQUEOUS
1
MICROBIAL
MUTAGENESIS,
CYTOTOXICITY
LIQUIDS
ORGANIC
1
f
WITH
SUSPENDED
SOLIDS
>
r
MICROBIAL MUTAGENESIS,
CYTOTOXICITY,
RODENT ACUTE TOXICITY,
ALGAL, FISH, AND
INVERTEBRATE BIOASSAY,
TERRESTRIAL BIOASSAYS1 2
>
f
MICROBIAL
MUTAGENESIS,
CYTOTOXICITY,
RODENT ACUTE
TOXICITY
SOLIDS
>
r
SAMPLE
>
z
>
i
-<
V.
CO
^ '
ALGAL
BIOASSAY,
FISH
BIOASSAY
INVERTEBRATE
BIOASSAY
TERRESTRIAL
BIOASSAYS1'2
1OPTIONAL BIOLOGICAL TESTS.
2TERRESTRIAL TESTS UNDER EVALUATION.
Figure 2. Biological analysis overview.
-------
LEVEL 1 CHEMICAL TESTS
The Level 1 sampling and analysis plan calls for simultaneous collection and
testing for biological and chemical parameters. The Level 1 chemical testing plan
uses spark source mass spectrometry (SSMS) for qualitative and semiquantitative
elemental analysis and ion chromatography (1C) for anion analysis. Gas
chromatography (GO, liquid chromatographic (LC) separation of organic com-
ponents into similar groups, gravimetric determinations, infrared spectroscopy (IR),
and low resolution mass spectrometry (LRMS) are employed for characterization of
organic compounds in a sample. As in the biological testing scheme, the Level 1
chemical testing scheme is designed to produce a semiquantitative and qualitative
characterization of a material. The more costly and time-consuming Level 2
chemical tests are quantitative and compound-specific, and these Level 2 tests are
employed where Level 1 tests indicate a potential for environmental detriment.
Figure 3, from the revised Level 1 Procedures Manual (reference 2), illustrates the
combined biological and chemical testing plan on solids, slurries, and liquids.
-------
LEACHABLE
MATERIALS
BiOASSAY
INORGANIC
SPECIES
ORGANIC
SPECIES
SUSPENDED
SOLIDS
BIOASSAY
INORGANIC
SPECIES
SELECTED
WATER
TESTS
(AQUEOUS)
ORGANIC
EXTRACTION
OR DIRECT
ANALYSIS
BIOASSAY
ORGANIC
SPECIES
INORGANIC
SPECIES
Elements and
selected anions
Physical separation
into fractions and
chemical classification
INORGANIC
Elements and
selected anions
ORGANIC
SPECIES
b.p. >100°C
ORGANIC
SPECIES
b.p. <100°C
Physical separation
into fractions and
chemical classification
Element: and
selected anions
^
SPECIES
ORGANIC
SPECIES
BIOASSAY
Elements and
selected anions
Physical separation
into fractions and
chemical classification
Physical separation
into fractions and
chemical classification
Aliquot for gas
chromitographic
analysis
Figure 3. Basic Level 1 sampling and analytical scheme for solids,
slurries, and liquids.
10
-------
SUMMARY AND RANKING OF THE RESULTS OF ENVIRONMENTAL ASSESSMENTS
The combined biological and chemical screening scheme produces a large number
of individual test results, and EPA/IERL-RTP has recognized a need to summarize these
results and numerically relate them to some degree of severity or hazard. In order to
summarize the chemical data, a list of threshold levels or minimal concentrations of
concern has been prepared for a large number of chemical compounds. This list is
generally based on whole animal tests of toxic materials and, more specifically, on
LD50 (lethal dose to 50 percent of a population) testing performed by oral administra-
tion of various substances to rodents (reference 3). If a particular chemical compound
or chemical group, in the worst case, exceeds the minimum level of concern, then
Level 2 testing is indicated.
A Source Assessment Model (SAM) (reference 4) has been devised for tabulating
the Level 1 chemical data. In this model, the Level 1 chemical data are formatted,
and chemical concentrations are ratioed to levels of concern. These ratios are to-
taled, giving discharge severities (DS). The rate of discharge of an effluent is fac-
tored in, and total discharge severity rates (DSR) are calculated.
Summarizing the biological data from such a wide variety of test types is a for-
midable task. Methods for introducing the complex biological data into this matrix of
information have recently been proposed by EPA/IERL-RTP (reference 5). These
methods are based on the maximum applicable dose (MAD) for each biological test,
which is the maximum amount of a substance that can be administered in a given
bioassay.
The MAD concept must be applied differently to each biological test. Response is
ranked as high, moderate, low, or nondetectable. Table 3 gives response ranges and
maximum applicable doses for several of the Level 1 bioassays.
In the Ames test, the criteria for a positive (mutagenic) response is a ratio of test
revertants to control revertants greater than 2 for Strains TA1535, TA1537,
TA1 538, TA98, or TA100, plus an increasing dose/response relationship over three
successive dose ranges.
11
-------
TABLE 3. RESPONSE RANGES FOR RANKING OF VARIOUS BIOTESTS
RESPONSE RANGES
ASSAY
Health Tests
Ames
RAM, CHO,WI-38
Rodent
Ecological Tests
Algae
Fish
Invertebrate
ACTIVITY MEASURED
Mutagenesis
Lethality (LC5(j)
Lethality (LD50)
Growth Inhibition (ECsg)
Lethality (LC50)
Lethality (LC50)
MAD
5 mg/plate or
500ML/plate
1,000 jUg/rnL or
600ML/mL
10 g/kg or
10mL/kg
1,OOOmg/L or
100%
1,000 mg/Lor
100%
1,000 mg/Lor
100%
HIGH
<0.05 mg or
<5ML
<10/zg or
<6ML
<0.1
<20%or
<200mg
<20% or
<200 mg
<20% or
<200 mg
MODERATE
0.05-0.5 mg or
5-50 ML
10-100 Mg or
6-60 ML
0.1-1.0
20-75% or
200-750 mg
20-75% or
200-750 mg
20-75% or
200-750 mg
LOW
0.5-5 mg or
50-500 ML
1 00-1,000 M9 or
60-600 ML
1-10
75-1 00% or
750-1,000 mg
75-1 00% or
750-1, 000 mg
75- 100% or
750-1, 000 mg
NOT DETECTABLE
ND at >5 mg or
NDat>500
LC5fj> 1,000 M9 or
LC5Q>600ML
LD5Q>10
ECgrj > 100% or
EC50>1,OOOmg
LC5g>100%or
LC5Q> 1,000 mg
LC5n>100%or
LC5Q> 1,000 mg
MAD = Maximum Applicable Dose (Technical Limitations)
= Calculated Dosage Expected to Kill 50% of Population
= Calculated Concentration Expected to Kill 50% of Population
= Calculated Concentration Expected to Produce Effect in 50% of Population
ND = Not Detectable
-------
CASE STUDIES: EXAMPLES OF LEVEL 1 OUTPUTS
Some examples of actual chemical and biological test results from Level 1 En-
vironmental Assessments illustrate Level 1 outputs and the applications of these
outputs. Three pilot studies were run to evaluate the Level 1 methodologies, and the
data shown here are drawn from these pilot studies. The studies involved
assessments of a coal gasifier, a series of textile plants' effluents, and a fluidized-
bed combustor (references 6, 7, and 8). Figure 4 shows the biological results for
samples chosen from the textile effluent pilot study to illustrate a range of biological
responses; this figure also shows a DS value for health and for ecology for each
sample. These DS values were calculated by summing the ratios of each chemical
test result to a threshold level of concern for that chemical category. Table 4
presents the health-related bioassay results from the pilot studies by decreasing DS-
health value, and Table 5 shows the ecologically related bioassay results ordered by
decreasing DS-ecological value. The most hazardous samples, based on chemical
analysis, are at the top of the lists. Because none of these samples caused a lethal
response in rats but some samples caused abnormal behavioral or physiological
responses, Table 4 also presents a sum of the number of abnormal responses.
Figure 5 shows how the bioassays can be used to rank samples; in this figure, all
samples are ranked for their cytotoxicity in RAM cells.
Several conclusions can be drawn from examination of the chemical and bioassay
results of these samples.
1. There was a total of 1 75 bioassays run on these 27 samples as shown in
Tables 4 and 5. The Level 1 bioassay screening procedures indicated that
110 of these (63 percent) showed no detectable toxicity. This screening
procedure eliminates the need for costly and time-consuming Level 2
bioassaying in many instances.
2. Relatively high chemically based health-related DS values usually occur in
samples with positive bioassays. Notable exceptions to this pattern do oc-
cur. For example, referring to Table 4, Plant X had a relatively low health-
related DS value but gave positive indication of cell toxicity in the RAM test.
3. There also is a correlation between the chemically derived ecological DS
values and ecologically related bioassays, but exceptions to this pattern of
interpretation also occur. For example, Plant W ranked 24th out of 27
samples for ecologically related, chemically based DS values, but Plant W
gave low or moderate responses on five of six ecological bioassays. Where
differences in the chemical model and the biological tests occur, the analysis
may not be measuring the same effect. Therefore, chemical tests and
biological assays complement each other and should be run in parallel.
4. The Discharge Severity Model, based on chemical measurements, shows a
fair correlation between ecologically related DS values and health-related DS
values. Those samples showing a high potential for health-related damage
also generally show a high potential for ecologically related damage.
5. The DS Model also shows a fair degree of agreement between health and
aquatic ecological tests; however, the ecological tests appear to be more
sensitive than the chemical model as presently performed for liquid samples.
13
-------
BIOLOGICAL
DISCHARGE
SEVERITY
CHEMICAL
DISCHARGE
SEVERITY
Severity
High
Moderate -
Low
PLANT W
I
DS
Log10
234 7 8 9 10 11 12
1 6
10
9
8
7
6
5
4
3
2
1
Severity
High
Moderate -
PLANTN
Low
DS
L°9io
10
9
8
7
6
5
4
3
2
234 7 8 9 10 11 12
1 6
Severity
High
Moderate
Low
PLANT X
N
N
N N N N N N
234
N = No detectable toxicity
DS = Discharge Severity
7 8 9 10 11 12
Health
DS
L°9lO
1 6
10
9
8
7
6
5
4
3
2
1
Ecological
1 = DS 6 = DS
2 = AMES 7 = FW ALGAL
3 = RAM 8 = FW FISH
4 RAT 9 = FWDAPHNIA
10 = SW ALGAL
11 = SW FISH
12 = SW GRASS SHRIMP
Figure 4. Case study of textile plants: selected biological and
chemical test results.
14
-------
VIABILITY INDEX
AT 1,000 fig/ml
100.-
DECREASING TOXICITY-
Figure 5. Ranking of samples by the RAM cytotoxicity bioassay.
15
-------
TABLE 4. HEALTH EFFECTS: SUMMARY OF CHEMICAL AND BIOLOGICAL TEST RESULTS
STUDY
Coal Gasifier
Fluidized-Bed Combustor
Coal Gasifier
Textile Effluent
Textile Effluent
Textile Effluent
Coal Gasifier
Textile Effluent
Fluidized-Bed Combustor
Textile Effluent
Fluidized-Bed Combustor
Textile Effluent
Fluidized-Bed Combustor
Textile Effluent
Textile Effluent
Textile Effluent
Fluidized-Bed Combustor
SAMPLE
Separator Liquor
Bed Reject Leachate
Separator Tar
Plant L
Plant W
Plant N
Gasifier Ash
Plant U
Fine Particulates
Plant F
Cyclone Dust
Plant B
Bed Reject
Plant G
Plant X
Plant E
Dolomite
1
DS
6.1 x irj7
1.2 x 106
1.0 X106
8.4 x 103
5.0 x 103
3.3 x 103
1.7 X103
8.9 x 102
7.0 x 102
4.5 x102
3.8 X102
3.3 X102
1.6 X102
9.4 X101
3.6 X101
3.3 X101
2.3 X101
2
AMES
N
*
H
N
N
N
N
N
L
N
N
N
N
N
N
N
N
3
RAM
M
N
L
M
L
M
N
N
L
L
N
N
N
N
L
N
N
4
RAT
N
#
*
N
N
N
N
N
#
N
#
N
#
#
N
N
*
5 RAT-
ABNORMAL
RESPONSES
0
*
*
5
0
6
0
0
*
4
*
6
*
#
0
0
*
* = Data Not Available
H = HIGHToxicity Rating
M = MODERATE Toxicity Rating
L = LOWToxicity Rating
N = No Detectable Toxicity
1 = DS = Discharge Severities
2 = AMES = Mutagenicity Test
3 = RAM = Rabbit Alveolar Macrophage
4 = RAT = Rodent Acute Toxicity
5 = RAT - Abnormal Responses = Sum of the Number of Sublethal
Abnormal Behavioral and Physiological Responses Noted
in the 10 Rats Used in Testing Each Sample
-------
TABLE 5. ECOLOGICAL EFFECTS: SUMMARY OF CHEMICAL AND BIOLOGICAL TEST RESULTS
STUDY
SAMPLE
Fluidized-Bed Combustor Bed Reject Leachate
Coal Gasifier
Fluidized-Bed Combustor
Coal Gasifier
Textile Effluent
Textile Effluent
Textile Effluent
Textile Effluent
Fluidized-Bed Combustor
Textile Effluent
Textile Effluent
Textile Effluent
Coal Gasifier
Textile Effluent
Fluidized-Bed Combustor
Fluidized-Bed Combustor
Textile Effluent
* = Data Not Available
H = HIGH Toxicity Rating
M = MODERATE Toxicity Rating
I = LOW Toxicity Rating
N = No Detectable Toxicity
Separator Liquor
Fine Particulates
Separator Tar
Plant N
Plant L
Plant B
Plant E
Cyclone Dust
Plant F
Plant U
Plant G
Gasifier Ash
Plant W
Bed Reject
Dolomite
Plant X
6
DS
1.
5
6.9
1.
2.
1.
6,
3,
2
0
9
.5
,1
.0
.9
2.6
2.
1,
1
9
5
2
4
3
.2
.5
.2
.3
.6
.7
.8
.4
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
108
107
106
10s
10s
104
104
104
104
104
104
104
103
103
103
102
102
7
FW
ALGAL
M
H
*
*
H
M
M
H
#
N
N
N
*
L
*
#
IM
6 =
7 =
8 =
9 =
10 =
11 =
12 =
8
FW
FISH
M
H
*
*
M
M
N
N
#
N
N
M
X-
M
*
#
N
9
FW
10
SW
DAPHNIA ALGAL
M
H
#
*
H
M
N
H
*
L
H
M
*
H
*
*
N
*
H
#
#
H
K
!\l
#
*-
L
N
M
*
M
*
#
N
11
SW
FISH
N
H
*
*
M
N
N
N
#
N
N
N
*
M
*
*
N
12
SW GRASS
SHRIMP
N
H
*
#
M
N
N
N
*
N
N
N
*
H
*
*
N
DS = Discharge Severities
FW ALGAL
FW FISH =
= Freshwater Algal
Fathead Minnow
FWDAPHNIA = Freshwater Invertebrate
SW ALGAL
SW FISH =
SW GRASS
= Marine Algal
Marine Sheepshead
Minnow
SHRIMP = Saltwater Shrimp
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CURRENT STATUS OF THE LEVEL 1 BIOLOGICAL SCREENING PROGRAM
The matrix of bioassays in Level 1 will continue to be reviewed and evaluated.
The following activities are being evaluated for possible inclusion in the screening
matrix:
Modifications to the rodent acute toxicity protocol to increase sensitivity.
Additional Level 1 screening tests for chronic health effects (i.e., car-
cinogenic).
Concentration and extraction methods for the Ames microbial mutagenesis
assay.
Comparison and selection of optimum cellular toxicity assays.
Evaluation and incorporation of soil, plant, and insect tests into the terrestrial
bioassay group.
Evaluation of chemical fractionation procedures compatible with bioassays.
Development of bioassays for testing gaseous samples.
Development of quality control procedures specific to Level 1 tests.
The health and ecological labs are also working closely with IERL-RTP on joint in-
terpretation of chemical/biological results.
BIOASSAY PROGRAMS IN OTHER EPA LABORATORIES
This cooperative biological testing program parallels the development of similar
programs in several other EPA laboratories and is consistent with more recent
development of similar programs within the Agency that stress multiple biological
endpoints in their test matrixes. The Office of Toxic Substances, the Office of
Pesticide Programs, the Office of Solid Wastes, the Environmental Monitoring and
Support Laboratory in Cincinnati (EMSL-Cin), and the Ocean Dumping Permits Of-
fice all require or propose to require bioassaying programs for the evaluation of
hazardous materials. Table 6 compares the Level 1 biological testing program with
the proposed or draft bioassay requirements of these other program offices.
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TABLE 6. COMPARISON OF IERL-RTP LEVEL 1 BIOASSAY
REQUIREMENTS WITH THOSE OF EPA PROGRAM OFFICES
BIOLOGICAL
TEST
In Vitro
Mutagenicity or
Carcinogenic! ty
In Vitro
Cytotoxicity
Whole Animal
Freshwater
Ecological
Marine
Ecological
Terrestrial
Ecological
IERL-RTP
LEVEL 1
Bacterial
Mutagenicity
Rabbit Alveolar
Macrophage
(For Participate)
WI-38orCHO
Cloning
(For Solutions)
Quanta!
Range Finding
Fathead Minnow
Algae
Daphnia Magna
Sheepshead Minnow
Algae
Grass Shrimp
Soil Respiration1"
Insect10
Root Elongation1"
Stress Ethylene
OFFICE OF
SOLID WASTE
Bacterial
Mutagenicity
Mammalian Somatic
Cells in Culture5
Fungal Microorganisms
DNA Repair in
Bacteria6
Unscheduled DNA
Repair in Human
Diploid Cells6
Mitotic Recombinatio
or Gene Conversion
in Yeast6
Sister-Chromatid
Exchange in
Mammalian Cells6
Daphnia
Reproduction
Germination
Seedling Growth
OFFICE OF TOXIC
SUBSTANCES
(ToSCA)1 -
Mutagenlc
Oncogenic
Subacute and
Chronic Toxicity
(6 Tests Suggested)
Epidemiological Studies
Teratogenic
Oncogenic
Reproductive
Germination
Seedling Growth
OCEAN DUMPING
PERMITS
Sheepshead Minnow9
Algae9
Grass Shrimp9
Mysid Shrimp9
Ascartia Tonsa9
EMSL-CIN (EPA
SUBCOMMITTEE)
Fathead Minnow
Algae
Daphnia
OFFICE OF
PESTICIDE PROGRAMS2 1
Bacterial
Mutagenicity*1
Eucaryotic Microorganism
Mutagenicity4
Mammalian Somatic Cells,
Mutagenicity4
Bacterial DNA Repair7
Mammalian Cells,
Unscheduled DNA Synthesis7
Yeast, Mitotic Recombination
or Gene Conversion7
Sister-Chromatid
Exchange7
Mammalian Cytogenetics8
Insect, Heritable Chromosomal Effects8
Rats, Dominant Lethal8
Rodents, Heritable Translocation8
Oncogenic (2 mammalian species)
Mouse, Specific Locus Test4
Insect, Sex-Linked Recessive Lethal4
1 The open wording of the Toxic Substances Control Act permits the possibility of including data from a wide variety of short-term tests as part of the test data requirements.
2 Efforts are underway to combine the requirements for bioassay for the Office of Toxic Substances and the Office of Pesticide Programs.
1 These tests have been proposed but are not yet legally required.
4 Proposed requirement of a minimum of three tests, but not more than one test of each type mentioned.
5 Choose one of the tests in this group to partially satisfy disposal requirements.
6 Choose two of the tests in this group to partially satisfy disposal requirements.
7 Proposed requirement of a minimum of two tests, but not more than one test of each type mentioned.
K Proposed requirement of a minimum of three tests, but not more than one test of each type mentioned.
" These tests are done in both short- and long-term modes.
" These tests are optional until validated-
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CONCLUSIONS
Chemical tests alone cannot adequately characterize the hazardous nature of a
complex material. A scarcity of adequate information on the biological effects of
pure compounds exists in many instances; and, even if such information were
available, the present level of knowledge on synergistic effects and antagonistic
effects is inadequate to predict the response of a biological system to an effluent or
emission composed of many chemical compounds. When high levels of a known
hazardous chemical compound are present in a sample, a strong biological response
can usually be anticipated, but the converse cannot be assumed. As illustrated in
the section on case studies, biological responses were noted in cases where
chemical DS rating was relatively low.
The present level of knowledge and the currently available information point
toward the cost-effective use of a Level 1 screening program that uses complemen-
tary bioassays and chemical tests for predicting environmental hazard. EPA/IERL-
RTP has used the described screening program in its ongoing environmental assess-
ment program with considerable success. Experience in the use of chemistry-
bioassay screening methodology common to many R&D programs has allowed
refinements of the tests to provide cost-efficient reliable assessments of en-
vironmental impact from a wide variety of source and sample types.
It should be reiterated that the health and ecological labs are working closely with
IERL-RTP on joint interpretation of chemical/biological results. The cooperative pro-
gram will continue to evaluate and revise the Level 1 biological protocols in order to
incorporate new developments in the area. For example, short-term subacute and
chronic tests applicable to Level 1 screening bioassays are being reviewed.
In addition, the subcommittee is in the process of structuring a Level 2 protocol
which will include subacute and chronic effects tests and/or fractionation of
samples for verification and quantification of results from the screening studies.
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REFERENCES
1. K. M. Duke, M. E. Davis, and A. J. Dennis, IERL-RTP Procedures Man-
ual: Level 1 Environmental Assessment Biological Tests for Pilot Studies,
EPA-600/7-77-043 (NTIS PB268484), April 1977.
2. D. E. Lentzen, D. E. Wagoner, E. D. Estes, and W. F. Gutknecht, IERL-RTP Pro-
cedures Manual: Level 1 Environmental Assessment (Second Edition), EPA-
600/7-78-201, October 1978.
3. J. G. Cleland and G. L. Kingsbury, Multimedia Environmental Goals for En-
vironmental Assessment, Vol. I and Vol. II, EPA-600/7-77-136a and EPA-
600/7-77-136b (NTIS PB276919 and 276920), November 1977.
4. L. M. Schalit and K. J. Wolfe, SAM-1A: A Rapid Screening Method for En-
vironmental Assessment of Fossil Energy Process Effluents, EPA-
600/7-78-015 (NTIS PB277088), February 1978.
5. D. Brusick and R. Hart, Internal Communication, Litton Bionetics, August
1978.
6. Radian Corporation, Environmental Assessment: Source Test and Evaluation
Report for a Commercial Chapman Low-Btu Gasification Facility, EPA-
600/7-78-202 (NTIS PB289940), October 1978.
7. G. D. Rawlings, Source Assessment: Textile Plant Wastewater Toxics Study
Phase I, EPA-600/2-78-004h (NTIS PB280959), March 1978.
8. J. M. Allen, K. M. Duke, Multimedia Emissions From Pressurized Fluidized Bed
Combustion of Coal, presented at the 71 st annual meeting of the Air Pollution
Control Association, Houston, Texas, June 25-30, 1978.
This paper was prepared by Research Triangle Institute under contract
#68-02-2688 with the Industrial Environmental Research Laboratory of the
Environmental Protection Agency.
21
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22
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/8-79-021
2.
4. TITLE AND SUBTITLE
Biological Screening of Complex Samples from
Industrial/Energy Processes
7. AUTHOR(S)
N.G. Sexton
9. PERFORMING ORGANIZATION NAME Al\
Research Triangle Institut
P.O. Box 12194
Research Triangle Park, 1
D ADDRESS
e
^orth Carolina 27709
12. SPONSORING AGENCY NAME AND ADDRESS
EPA, Office of Research and Development
Industrial Environmental Research Laboratory
Research Triangle Park, NC 27711
3. RECIPIENT'S ACCESSION NO.
5. REPORT DATE
August 1979
6. PERFORMING ORGANIZATION CODE
8. PERFORMING ORGANIZATION REPORT NO.
10. PROGRAM ELEMENT NO.
1AB604 and 1BB610
11. CONTRACT/GRANT NO.
68-02-2688, Task 104
13. TYPE OF REPORT AND P,ERIOD COVERED
Special; 9/78 - 7/79
14. SPONSORING AGENCY CODE
EPA/600/13
15. SUPPLEMENTARY NOTES ffiRL-RTP project officer is Raymond G. Merrill, Mail Drop 62,
919/541-2557.
16. ABSTRACT
The report describes a cooperative project between EPA/IERL-RTP and EPA
health and ecological laboratories to establish a biological screening program for
complex samples from industrial and energy processes. The report summarizes
the program elements and their application to various complex environmental sam-
ples. Results from the application of this program show that it is effective for
screening complex mixtures. The report also describes the data formatting proce-
dures used to report results from a diversity of biological tests in a meaningful way.
It also compares the biological screening approach to proposed programs from
other EPA program offices: the comparison shows a similarity in the approach for
biological screening within EPA.
17.
a. DESCRIPTORS
Pollution
Bioassay
Tests
Industrial Processes
Energy Processes
13. DISTRIBUTION STATEMENT
Release to Public
KEY WORDS AND DOCUMENT ANALYSIS
b. IDENTIFIERS/OPEN ENDED TERMS
Pollution Control
Stationary Sources
Biological Screening
19. SECURITY CLASS (This Report)
Unclassified
20. SECURITY CLASS (This page)
Unclassified
c. COSATI Field/Group
13B
06A
14B
13H
10B
21. NO. OF PAGES
24
22. PRICE
EPA Form 2220-1 (9-73)
23
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