United States
Environmental Protection
Agency
Air and Energy Engineering
Research Laboratory
Research Triangle Park NC 27711
Research and Development
EPA/600/S8-86/029 Jan. 1987
Project Summary
AEERL Procedures Manual:
Level 1 Environmental
Assessment Terrestrial
Ecological Tests
Thomas E. Gezo and David J. Brusick
Some test methods designated for
EPA/AEERL's Level 1 environmental as-
sessment biological testing program are
sufficiently new that little or no published
literature is available describing specific
application of the procedures. This manual
provides detailed procedures for EPA/
AEERL's Level 1 terrestrial bioassays. The
manual supplements the "IERL-RTP Pro-
cedures Manual: Level 1 Environmental
Assessment Biological Tests" (second edi-
tion, EPA-600/8-81-024, NTIS PB
82-228966) which provides the basic pro-
cedural steps. The protocols described in
this manual are the same as those in the
supplemented manual, but are expanded
in those areas of methodology which in-
volve culturing methods, materials and
supplies, and specific scoring procedures.
The three tests included in this manual
are: (a) Plant Stress Ethylene Test, to
assess the toxic effects of gaseous sam-
ples on plant foliage; (b) Root Elongation
Test, to determine the toxic effects of
liquid samples and aqueous leachates of
solid samples on seed germination and
root elongation in a number of plant spe-
cies; and (c) Insect Toxicity Test, to mea-
sure lethality and reproductive toxicity in
Drosophila melanogasterto both liquid and
some solid samples.
This Project Summary was developed
by EPA's Air and Energy Engineering Re-
search Laboratory, Research Triangle Park,
NC, to announce key findings of the re-
search project that is fully documented in
a separate report of the same title (see
Project Report ordering information at
bade).
Introduction
This bioassay manual supplements EPA/
AEERL's latest procedures manual for Level
1 environmental assessment biological
tests.* There is little published material on
the application of the Level 1 terrestrial
bioassays for environmental assessments.
Thus, this manual provides a more detailed
description of the terrestrial bioassays
than are presented in the Level 1 biological
testing manual. The protocols described
in this manual are the same as those in the
supplemented manual. Details and refer-
ences to assist laboratories in implement-
ing Level 1 terrestrial ecological effects
testing have been added.
The Level 1 terrestrial ecological effects
tests include assays for determining tox-
icity of complex industrial process streams
in plant and insect test organisms. The
tests are able to detect sublethal toxic
response to stress in plants (Plant Stress
Ethylene Test—PSE), sublethal and lethal
toxic responses in germinating seeds
(Root Elongation Test—RE), and acute tox-
icity and reproductive impairment in in-
sects (Insect Toxicity Test—IT).
These tests provide a range of terrestrial
organisms for assessing the relative effect
of effluent streams on the environment.
Test organisms include maturing plants,
germinating seeds, and insects. These
tests offer testing capabilities for all sam-
"Brusick, D.J., and R.R. Young. "IERL-RTP Procedures
Manual: Level 1 Environmental Assessment
Biological Tests," EPA-600/8-81-024, NTIS No. PB
82-228966, Litton Bionetics, Inc., Kensington, MD,
October 1981, 150 pp.
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pie types (including gases) with the advan-
tages of low cost, reproducibility, and
relatively rapid performance. A future goal
for the Level 1 program is to include a test
procedure for assessing the impact of ef-
fluent samples on soil microorganisms
(decomposers).
Terrestrial ecological tests are used to
determine the concentration of test mate-
rial that produces a defined toxic effect on
a specified percentage of the test organ-
isms in a fixed period of time. The PSE test
is designed to assess and rank the toxic
effects of gaseous effluents on plants by
measuring the stress ethylene of plant
response and by assessing relative foliar
injury in exposed plants. The RE test
measures the inhibition of root elongation
and seed germination; although both are
observable toxic responses and are re-
ported, root-elongation inhibition is the
preferred end point. The concentration
which inhibits root elongation by 50% of
the control (ECSO) is estimated and used
to rank effluent samples. The IT assay
measures the acute toxicity and reproduc-
tive capacity of fruit flies treated with
environmental samples. The dose lethal to
50% of the flies (LD50) compared to the
control is calculated and used to rank test
samples. In the optional fertility test, the
effective concentration which reduces the
fecundity of surviving dosed flies to 50%
of control flies EC50 is calculated.
Characteristics of the Level 1 terrestrial
bioassays are given in Table 1.
Level 1 Plant Stress Ethylene (PSE)
Test
The EPA Level 1 PSE test is designed to
assess the toxic effects of gaseous efflu-
ents on plants by employing the stress
ethylene plant response. Under normal
conditions plants release low levels of
ethylene which function hormonally to
regulate growth and development. In
response to various stresses, ethylene pro-
duction increases substantially. Viable
plant tissue is necessary for ethylene
production. Ethane evolution may also
increase in response to some stresses and
is thought to indicate more severe damage
and tissue autolysis. Critical to the effec-
tiveness and applicability of the PSE test
is that induction of stress ethylene is pro-
portional to the intensity and duration of
the stress over a wide range of stresses.
However, the stress ethylene response has
yet to be documented for a large number
of pollutants and complex mixtures.
The test procedure minimizes laboratory
manipulation to provide a cost effective
bioassay. The procedure involves culturing
Table 1. Characteristics of Level 1 Terrestrial
Ecological Effects Bioassays
Characteristic
Species
End point (s)
measured
Amenable to
sample type
Data expression
Special features
Plant Stress
Ethylene Test
Bush bean
Metabolic stress
evidenced by
ethylene production,
foliar damage
Gases
Positive or negative
Only validated
Level 1 bioassay
for gases; sensitive
Root Elongation
Test
Cucumber, wheat,
red clover,
radish, lettuce
Root length,
germination
Liquids, solids
(leachatesi
EC50
Detects toxi-
city to terres-
trial producers;
multiple species
Insect Toxi-
city Test
Drosophila
melanogaster
Lethality,
reproductive
capacity
Liquids,
solids
i-Dso
Detects lethality
to terrestrial con-
sumer; can a/so pro-
vide data on fertility
of test plants, collection of gas samples,
exposure, incubation, ethylene analysis,
and visual examination. Bush bean,
Phaseolus vulgaris L., cultivar Harvester,
has been selected as the bioassay plant
because it develops rapidly and is easy to
grow. The plants are cultured under con-
trolled conditions and are subsequently ex-
posed to the test or control (chlorine) gas.
After a period of dark incubation in sealed
containers, ethylene and ethane are anal-
yzed by gas chromatography. Three days
after the assay, the plants are examined
visually for injury.
Chapter 1 of the full report contains de-
tailed procedures for gas sampling, design
and construction of exposure chambers,
and methods for exposing test plants to
control and test gases. Also discussed in
detail are plant culturing techniques, post-
exposure incubation, and ethylene and
ethane analysis. Visual inspection of the
test plants is also discussed as an adjunct
method for assessing test material toxic-
ity. Finally, criteria for assay acceptance,
interpretation, and evaluation are
presented.
Level 1 Root Elongation (RE) Test
Assessing phytotoxic effects requires
the selection of a stage in plant develop-
ment that is sensitive to a broad range of
toxicants and is important physiologically.
Seed germination and root elongation, crit-
ical links in plant development between a
dormant embryo and a period of rapid
growth with the formation of essential
plant structures, were selected for phyto-
toxicity testing. Toxic substances that pre-
vent or reduce germination or root elonga-
tion will decrease plant populations and
reduce crop yields. In natural systems,
affected species are less able to compete
with other species, and tolerant specie
may be selected, resulting in changes ii
species diversity, numbers, and popule
tions dynamics.
The RE/seed germination bioassay, i
Chapter 2 of the full report, is a rapid an
simple test. Germination and root elongc
tion can be observed after 115 hours of ir
cubation, and the test does not requir
large expenditures for equipment and fc
cilities or complicated techniques. The R
test is suitable for testing liquid sample
or solid samples from which aqueou
leachates can be prepared.
A toxicant may cause responses at di
ferent doses in different plant species."
detect an effect from chemicals of ui
known toxicity, several plant species a
selected. The species used in this test a
lettuce (Butter Crunch), Lactuca sativa I
radish (Cherry Belle), Raphanus sativus
wheat (Stephens), Triticum aestivum
cucumber (Hybrid Spartan Valo
Cucumis sativus L.; and red clo\
(Kenland), Trifolium pratense L. The
species are representative of economic?
important plants and different pi;
families. The seeds chosen germinate i
grow rapidly, contain no natural inhibitc
and require no special pretreatment.
test organisms are grown under ident
environmental conditions (constant 2E
temperature constant dark, and enclo
to maintain uniformly high relative hur
ity) and are exposed to several effh
concentrations. Although inhibitior
both RE and germination are observ
toxic responses, in this bioassay RE ir
tion is the preferred end point. Usuall
is inhibited at lower concentrations of
ic substances than is seed germirtc
Testing approaches are described,
criteria for interpretation are includ
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Chapter 2 of the full report gives a de-
ailed description of the methodology for
he RE test. General procedures are dis-
cussed concerning the test organisms,
test chambers, reagent and material prep-
ration, and the preparation of test material
and control solutions. The procedures for
planting the seeds, exposure to test solu-
tions, incubation, and data collection in
both range-finding and definitive tests are
also presented. The assay acceptance, in-
terpretation, and evaluation criteria are
discussed in detail.
Level 1 Insect Toxicity (IT)
Bioassay
Drosophila melanogaster is a common
insect species in nature; many features of
this organism make it attractive as an in
vivo test system for detecting environ-
mental toxicants. These features include:
1) a short life cycle of 10-12 days; 2)
minimal space, monetary, and manpower
requirements to maintain stocks; 3) the
ability to detect toxic effects at specific
life cycle stages (adult, germinal, and
developmental); 4) a well-defined genetic
system which makes the detection of
specific genetic end points possible; 5) the
ability to biotransform genotoxic chemi-
cals via in vivo metabolic enzyme systems;
and 6) amenability to exposure to liquids,
soluble solids, gasses, and small particles.
-eeding via solution is the most common
route of exposure.
Drosophila is included in EPA Level 1 en-
vironmental assessment testing not only
because of its application as a toxicity
screening test, but also because it can be
expanded into testing for additional end
points. For Level 1 applications, Drosophila
will be used to assess acute toxicity in the
adult flies and reproductive capacity
among surviving flies following exposure
to, or treatment with, environmental sam-
ples. The acute toxicity data will be used
to rank effluent streams and identify
sources for further testing. The fertility
response will be recorded and may be used
to provide possible direction for additional
testing.
Details of the Level 1 IT protocol are pre-
sented in Chapter 3 of the full report. The
source of and culture methods for Droso-
phila are presented. The methods to per-
form a basic feeding study are discussed.
The design of range-finding tests, defini-
tive tests for LD50 determination, and fer-
tility tests are presented as modifications
of the basic feeding-study test design.
Methods for data collection and interpreta-
tion and the criteria for test material rank-
ing are also included in Chapter 3.
T. A. Gezo is with Union Carbide Corp., Tarrytown, NY 10591; andD. J. Brusick is
with Litton Bionetics, Inc.. Kensington, MD 20895.
Raymond G. Merrill is the EPA Project Officer (see below).
The complete report, entitled "AEERL Procedures Manual: Level 1 Environmental
Assessment Terrestrial Ecological Tests, "(Order No. PB 87-102 398'/AS; Cost:
$11.95, 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:
Air and Energy Engineering Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
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