S-EPA
United States
Environmental Protection
Agency
Environmental Research
Laboratory
Cor vail is OR 97333
Research and Development EPA-600/D-82-274 August 1982
ENVIRONMENTAL
RESEARCH BRIEF
Development of a Test-Tube Stress-Ethylene
Bioassay for Detecting Phytotoxic Air Pollutants
L E. Craker
University of Massachusetts, Amherst, MA 01003
Abstract
The primary purpose of this study was to develop stress-
ethylene production from plant seedlings as a simple, rapid
and quantitative bioassay for detecting phytotoxic air pollu-
tants. The developed procedure was to require only small
quantities of gas for phytotoxic testing, have minimum cul-
tural and space needs, be easily standardized for comparison
of results from diverse testing laboratories, and provide an
unbiased estimate of phytotoxic activity.
Wheat, Triticum aestivum L, and tomato, Lycopersicon
esculentum L., seedlings growing on 3 ml of a defined agar
nutrient media in test tubes were evaluated for their stress-
ethylene production following exposure to phytotoxic air
pollutants. The test tubes containing the seedlings served as
both the pollutant exposure chamber and container for col-
lection of stress-ethylene. Sensitivity of the seedling tissue
to phytotoxic air pollutants and the presence of phyto-
toxicants in an air sample were indicated by increased
ethylene evolution from seedlings exposed to pollutants as
compared with control seedlings not exposed to pollutants.
Applicability of the bioassay was determined by testing five
known phytotoxicants: sulfur dioxide, nitrogen dioxide,
chlorine, hydrogen sulfide, and ozone. Except for ozone,
seedling tissue was treated with the pollutant by sealing the
top of the test tube with a rubber serum cap and injecting the
pollutant through the serum cap with a hypodermic needle
and syringe. Seedlings were exposed to ozone by placing
the test tube (not sealed with serum cap) into a large ozone
treatment chamber. Stress-ethylene production was mea-
sured by gas chromatography of a 2-ml gas sample removed
from each tube.
Results indicated that stress-ethylene production from
wheat or tomato seedlings could be used to indicate the
presence of a phytotoxic pollutant. Since the test plants
were grown on an agar medium in test tubes, the procedure
could be replicated a large number of times in a matter of
hours, requiring only a small amount of air pollutant and a
minimum amount of space. The measurement of stress-
ethylene provided an unbiased estimate of phytotoxic activity.
Introduction
In order to establish safe release guidelines, government
agencies and industrial laboratories need a rapid, simple and
accurate means of screening smokestack emissions, indus-
trial compounds, and other air samples for phytotoxicants.
Current air pollution screening bioassays generally involve
large commitments of time and facilities because of relatively
long-term growth periods required for test plants plus large
quantities of pollutant and special fumigation chambers for
exposing plants to pollutants. In addition, the injury patterns
induced by phytotoxic substances can vary from species to
species and from pollutant to pollutant, making visible as-
sessment of air pollution subject to human bias.
The bioassay in this report is based on knowledge that injured
or stressed plants produce ethylene, that the level of ethy-
lene production by plants is generally related to the level of
injury or stress, and that ethylene can be quantitatively ana-
lyzed with a gas chromatograph. Plant seedlings were
chosen as the test organisms since they would have simple
environmental and nutrient requirements and may be grown
in large numbers in a small amount of space. Growth of the
seedlings in test tubes allowed the use of the tubes as both
the air pollutant treatment chamber and the container for
stress-ethylene collection.
A defined air pollutant bioassay protocol was to be devel-
oped by testing the suitability of plant seedlings in a stress-
ethylene bioassay and determining the optimal growth and
treatment/response conditions for maximizing the sensitiv-
ity of the bioassay. Both phytotoxic and non-phytotoxic
gases were to be tested to determine the accuracy of the
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bioassay. Different concentrations of pollutants were to be
tested to define the relationship between pollutant concen-
tration and stress-ethylene production.
Experimental
Plant Material
Wheat, Triticum aestivum L, and tomato, Lycopersicon
esculentum L, seedlings were selected for these studies
because they could be easily grown in test tubes on a simple
agar growth media and the plants have been shown to be
susceptible to air pollutant injury. Experimental testing was
done primarily with the wheat cultivar Olaf and the tomato
cultivar Roma VF, although other varieties and cultivars
were also tested. A total of 2 7 varieties of wheat were eval-
uated for ozone sensitivity by measuring stress-ethylene
production.
To initiate uniform germination and growth, the wheat seeds
were cold stratified for five days at 5 ° and the tomato seeds
were imbibed in distilled water at 25 ° for 24 hours before
planting. Seeds were subsequently surface sterilized in a
1 % sodium hypochlorite solution for 10 minutes, rinsed
with distilled water and individually planted in 23 ml (1.5 x
1 5 cm) test tubes containing 3 ml of sterilized agar growth
medium [1% agar and half-strength Hoagland's solution
(Hoagland and Arnon, 1 950) (Table 1)]. Following planting,
test tubes containing seeds were stoppered with cotton
plugs to prevent airborne pathogenic contamination and
placed into controlled environment chambers for germina-
tion and growth [ 1 6 hr day/8 hr night cycle; 65^Em-2s-1
(400-700 nm); with a constant temperature of 18° for
wheat, 27 ° for tomato]. To provide for water loss and to
maintain stomatal openings, 0.5 ml of half-strength Hoag-
land's solution was supplied to wheat plants every five days
and to tomato plants the day before exposure to pollutants.
Air Pollutant Treatments
Sulfur dioxide, chlorine, hydrogen sulfide, nitrogen dioxide,
and ozone were selected for studying stress-ethylene pro-
duction by wheat and tomato seedlings because of their
known phytotoxicity to plants. Concentrations of pollutant
tested were selected by determining the minimum amount
of pollutant that would initiate stress-ethylene production
by the seedlings during the short exposure period'.
Exposure of plant tissue to all air pollutants except ozone
was accomplished by replacing the cotton plugs of each test
tube with a rubber serum cap and injecting pollutant into the
sealed tube with a syringe and needle to produce a known
Table 1. Agar Growth Medium for Growing Test Plants
Ingredient
Agar
Ca(N03)2 •
KNO3
MgSC-4 •
KH2P04
H3B03
MnCI2
ZnS04
CuS04
H2MoO4 •
NaFe EDTA
4H
7H
4H
7H
5H
2
2
2
2
2
0
0
0
0
0
H2O
Concentration
10
1
9
1
.0
.590
.253
.247
.068
.43 x
.05 x
.1 x
4.0 x
1
3
.0 x
.94 x
10
10
10
10
10
10
(g/n
-3
-4
-4
-5
-5
-3
concentration. Each test tube was then returned to the same
controlled environment chamber used to grow the seedlings
for an exposure/collection period during which the pollutant
interacted with the seedling and the stress-ethylene produc-
ed by the seedlings in response to the pollutant treatment
was trapped in the sealed test tube.
Seedlings treated with ozone had the cotton plugs removed
and were then placed in a Plexiglas treatment chamber (102
cm x 58 cm x 36 cm). Ozone, generated by ultraviolet
light, was pumped into the treatment chamber as necessary
to maintain a fixed level of ozone for treatment of the seedlings.
At the end of the treatment period, the test tubes containing
the treated seedlings were sealed with rubber serum caps
and replaced in a controlled environment chamber for collec-
tion of stress-ethylene.
Stress-Ethylene Measurement
At the end of the exposure/collection period or collection
period (ozone), a 2-ml gas sample was removed from each
test tube and quantitatively analyzed for ethylene using a
gas chromatograph (sensitive to five ppb ethylene). Following
ethylene analysis, fresh weight of the top growth from each
plant was determined. Control seedlings were grown the
same as pollutant-treated seedlings, except there was no
pollutant treatment. Differences in ethylene production (ex-
pressed on a per unit fresh weight of leaf tissue basis) be-
tween pollutant-treated plants and control plants indicated
the stress-ethylene resulting from pollutant exposure.
The optimum age at which the seedlings could be effectively
used to test for phytotoxicity was determined by exposing
different age seedlings to pollutants for two hours. The most
effective length of the exposure/collection period was deter-
mined by monitoring ethylene production from seedlings ex-
posed to pollutants for different lengths of time.
All tests were replicated a minimum of three times with five
samples per treatment per replicate. Differences in amounts
of ethylene produced between treated and control seedlings,
differences among age of seedlings and differences among
exposure and collection periods were tested for significance
by analysis of variance. Ethylene production by wheat and
tomato seedlings in response to various concentrations of
phytotoxicants was examined by regression analysis.
Results and Discussion
Measurable increases in ethylene production were observed
in both wheat (Table 2) and tomato (Table 3) seedlings ex-
posed to phytotoxic air pollutants. The amount of ethylene
Table 2. Stress-Ethylene Production by Olaf Wheat
Seedlings Exposed to Air Pollutants.
The wheat seedlings were grown on an agar
medium in test tubes for six days and then
exposed to indicated concentrations of
pollutants for 2 hr. Mean ± S.E.
Ethylene Production
Pollutant nl/g fresh wt • 2 hr % above control
CI2 @ 100^1/1
03 @ 0.35^1/1
S02 @ 1 00 \AI\
NO2 @ 100^1/1
H2S @ 1 00 nl/l
CO @ 200 p
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produced varied with both the pollutant and the plant species.
Wheat plants treated with 200 ^I/liter of carbon monoxide (a
non-phytotoxic gas except at very high concentrations) did
not stimulate ethylene production above that of control
plants.
There was an effect of seedling age on the synthesis of
ethylene by the plant tissue (Figure 1). Significant differences
in ethylene production between seedlings exposed to phyto-
toxic air pollutants and controls were observed in wheat at
Table 3. Stress-Ethylene Production by Roma VF Tomato
Seedlings Exposed to Air Pollutants. The
tomato seedlings were grown on an agar
medium in test tubes for six days and then
exposed to indicated concentrations of
pollutants for 2 hours. Mean ± S.E.
Ethylene Production
Pollutant
nl/g fresh wt • 2 hr
above control
CI2 @ 1 00 Ml/I
S02 @ 1 00 Ml/I
N02 @ 400 Ml/I
H2S @ 400 Ml/I
Control
4.49 ± 0.07
2.55 ± 0.47
2.43 ± 0.17
2.81 ± 0.50
1.53 ± 0.21
193
67
59
84
0
10.0-
77 3 4 5 6 7 8 9 10 11 12 13 14 IS 16
Seedling Age, days
Figure 1. Seedling age and stress-ethylene production. Wheat
and tomato seedlings, grown on an agar medium in
test-tubes for indicated days, were exposed to 0.35
Ail/l O3 or 100//I/I CI2, respectively, for 2 hours
(Treated) or not treated with a pollutant (Control).
all seedling ages tested and in tomato at all seedling ages
tested except for five days.
The constancy of ethylene production by wheat seedlings
ranging in age from 5 to 16 days from seeding and by tomato
seedlings ranging in age from 9 to 1 6 days from seeding,
suggested that any age seedling within these ranges could
be used in a stress-ethylene test for phytotoxicants. How-
ever, there were limitations created by the size of the test
tube. After 8 days in wheat and 12 days in tomato, the seed-
lings had grown so large that there was a physical restriction
on continued leaf expansion in tomato due to the sides of the
tube and in wheat due to the cotton stopper at the top of the
tube. In the case of wheat over 8 days old, the tissue was
easily injured when the rubber serum cap was placed in the
top of the test tube. To avoid problems of increased ethylene
production due to growing and mechanical stresses, wheat
plants less than 8 days old and tomato plants less than 12
days old should be used in the test procedure.
The relatively large amounts of ethylene produced in both
the pollutant-treated and control seedlings at early ages (3
to 4 days in wheat; 5 to 8 days in tomato) were associated
with the early development of the plant tissue. To insure
that all seedlings had reached the stage of development
where ethylene was produced at a constant level, seedlings
older than 5 days in wheat and 9 days in tomato should be
used. We recommend using 6 day old wheat and 1 1 day old
tomato seedlings.
Our results indicated an exposure/collection period of two
hours was adequate for both inducing the plant tissue to pro-
duce ethylene and for collection of sufficient ethylene for
measurement by gas chromatography (Figure 2). Although
differences in ethylene production between pollutant-treated
and control plants could be measured at exposure/collection
6
Exposure/Collection Period, hour
Figure 2. Time course of stress-ethylene production. Tomato
seedlings, grown on an agar medium in test-tubes for
11 days, were either exposed to 40 /j\/\ CI2 for
indicated times (Treated) or not treated with a pollut-
ant (Control). Vertical lines indicate S.E.
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Table 4. Relationship of Ethylene Production to Pollutant Concentration
Plant
Species
Wheat
Tomato
Pollutant and
Range Tested (^l/l)
CI2, 0-1 OOc
SO2, 0-1 OOc
N02, 0-400d
H2S, 0-400d
CI2, 0-1 00C
S02, 0-1 00C
N02, 0-400d
H2S, 0-400d
Linear Response
Range (/jl/l)
0-80
0-100
0-400
0-400
0-40
20-100
0-400
0-300
Regression Equation
for Linear Response3
Y = 2.00 + 0.044X
Y = 1.09 + 0.01 3X
Y = 1.41 + 0.004X
Y = 1.41 + 0.004X
Y = 1.86 + 0.045X
Y = 0.68 + 0.022X
Y = 1.21 + 0.002X
Y = 1.73 + 0.004X
r2>
0.90
0.90
0.90
0.90
0.90
0.94
0.98
0.79
aAII regression coefficients were significant at P< 0.001.
bCoefficient of determination.
cConcentrations of pollutant increased in units of 20 n\/\.
dConcentrations of pollutant increased in units of 100 n\/\.
periods less than two hours, low concentrations of phyto-
toxic gases did not induce the production of enough stress-
ethylene for distinguishing between control and pollutant-
treated seedlings. Any collection period with a duration of 2
to 8 hours (longest exposure/collection period studied)
would be satisfactory, since the magnitude of difference in
ethylene production between treated and control seedlings
was maintained. However, the two-hour exposure/collection
period was sufficient and allowed for completion of the phy-
totoxicity test within one working day.
The amount of ethylene produced by pollutant-treated wheat
and tomato seedlings was related to the concentration of
the pollutant to which the plants were exposed. Based on
this relationship, it appears that the magnitude of pollutant
concentration within an air sample can be evaluated by the
amount of stress-ethylene produced (Table 4, Figure 3).
For all the phytotoxic gases tested, there was a range of pol-
lutant concentrations where an increase in concentration
initiated an increase in ethylene production. Concentrations
above this range gave only small or no further increases in
production of stress-ethylene by the plant tissue, depending
upon the pollutant.
Significant differences in stress-ethylene production follow-
ing exposures to ozone were observed in wheat cultivars
(Table 5). These differences suggest a test-tube stress-
ethylene bioassay may be capable of distinguishing plant
sensitivity to air pollutants.
The development of this bioassay has minimized the quantity
of test gas necessary for a phytotoxicity test and reduced
the need for greenhouse and fumigation facilities. The rec-
ommended procedure for a test-tube stress-ethylene bio-
assay (TTSEB) is presented in Table 6. Positive phytotoxic
responses to SO2 have been observed in tests conducted
over 18 months. The bioassay developed in this proposal
should provide a relatively inexpensive method of rapidly
screening large numbers of air pollutants for phytotoxicity
activity by both government agencies and private industry.
40
Si 3-°
I?
£5 20
e ®
.|»
fi= 1 0
0.1 0.2 0.3
Ozone Concentration,
//I/I 2 hr
0.4
Figure 3.
The effect of ozone concentration on ethylene
production by wheat seedlings. Olaf wheat seedlings
were grown on an agar medium in test-tubes for 6
days and then exposed to indicated concentration of
ozone for 2 hours. Each point represents the mean +
S.E. of 4 replicates (Grant, 1980).
Literature Cited
Grant, L. 1 980. The effectiveness of stress-ethylene as a
screening technique for a varietal susceptibility to air pollu-
tion. M.S. Thesis, Dept. of Plant and Soil Science, University
of Massachusetts, Amherst, MA 01003.
Hoagland, D.R. and D.I. Arnon. 1950. The water culture
method for growing plants without soil. Circular 347. Uni-
versity of California Experiment Station Bulletin.
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Table 5. Sensitivity of Wheat Cultivars to Ozone as
Indicated by Stress-Ethylene Production
(Grant, 1980)
Stress-Ethylene Production1
Cultivar (nl/g Fwt • 2 hr)
Waldron*2
Coteau*
Coteau*
Olaf*
Butte *
Olaf*
Prodax*
Olaf*
Fortuna*
Newana*
Ellar
Newana*
Eureka
Butte*
WS25
Chris
Waldron*
Solar
Parker 76
Triumph
Kitt
Prodax*
Protor
Profit 75
Bounty 309
Eagle
Centurk
Era*
Fortuna*
Newton
WS 1 809
Angus
Lew
Era*
James
Funk W444
1 Difference
Each value
2 Asterisks ir
0.37 a3
0.43 a
0.56 be
0.64 cd
0.70 d
0.73 d
0.73 d
0.74d
0.82 ef
0.86 fg
0.89 fgh
0.90 gh
0.95 hi
0.99 ij
1.04jk
1.05 jk
1.11 kl
1.13 I
1.16 I
1.16 I
1.25 m
1.25 m
1.29 mn
1.36 no
1.37 o
1.39 o
1.41 o
1.51 p
1.55 pq
1 .56 pq
1.63 qr
1.69 r
1.88 st
1.92 t
2.08 u
2.84 v
between control and ozone-treated seedlings.
represents the mean of 4 replicates.
idicate that more than nnf> sand snurra nf this
Table 6. Procedures Used In Test-Tube Stress-Ethylene
Bioassay (TTSEB)
1. Prepare sterile growth medium in 23 ml test tubes (3
ml/tube).
2. Plant individual seeds of wheat in each test tube contain-
ing growth media and stopper tubes with sterile cotton
plugs.
3. Allow seeds to germinate and seedlings to develop.
4. At 5 days from seeding, add 0.5 ml of water to each tube
to prevent water stress.
5. At 6 days from seeding, seal test tubes containing wheat
seedlings with rubber vaccine caps and add air sample
containing suspected phytotoxicant(s) through vaccine
cap with hypodermic syringe and needle.
6. Allow 2 hours for exposure of plant to phytotoxicant(s)
and collection of stress-ethylene in sealed tube.
7. Remove gas sample from each tube with hypodermic syr-
inge and needle and quantitatively analyze for ethylene
by gas chromatography.
8. Determine vegetative fresh weight of each seedling.
9. Express stress-ethylene production for each seedling as
nl/g FWT • 2 hr and compare with ethylene production
from wheat seedlings not exposed to phytotoxicant.
cultivar was tested.
3 Mean separation within cultivars by Duncan's new multi-
ple range test. Values followed by the same letter are not
significantly different (P < 0.05).
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