&ER&
United States
Environmental Protection
Agency
Environmental Research
Laboratory
Corvallis OR 97330
Research and Development
EPA-600/S3-81-006 May 1981
Project Summary
Development of a Protocol for
Testing Effects of Toxic
Substances on Plants
C. Ray Thompson, Gerrit Kats, Philip Dawson, and Denise Doyle
The purpose of this study was to
devise a rapid, simple, and reproducible
bioassay procedure to determine the
effects of toxic substances on vegeta-
tion and to provide a standardized
procedure for evaluating and compar-
ing the effects of diverse compounds.
Eight different plant species were
evaluated for rapid production of leaf
tissue, uniformity within the particular
cultivar, structural characteristics,
and potential for high ethylene pro-
duction when exposed to mild stress.
The plants were grown in a growth
chamber in small plastic pots in a
commercial potting mix, beans for 9
to 10 days and cucumbers for 14 days
prior to spraying. A photoperiod of 12
hours produced plants which evolved
the most stress ethylene. The test
compounds were applied with a modi-
fied pendulum sprayer equipped to
spray a single plant placed beneath the
center of its arc of swing. Prior to
spraying, the plants were exposed to
light for two hours. Thirty minutes
after spraying, the plants were encap-
sulated under half gallon glass jars
with a water seal, and were incubated
for 24 hours in a dark chamber at
°C. Five dosages of test compounds
were used with 8 replicates for the
final evaluation.
Ethylene samples were removed
from the jars with a syringe having a
bent needle. Concentrations of ethylene
were determined with a calibrated
Aerograph 1520 gas chromatograph.
A computer was used to plot the
stress-ethylene evolved from plants
versus the amount of compound ap-
plied from the equation: Loga (ethylene
concentration) = Loge A+ B (concentra-
tion of the toxicant).
Seven compounds were tested: two
organic herbicides (Paraquat and En-
dothall), three inorganic plant toxi-
cants (Phytar, sodium fluoride and
sodium chlorate), and two insecticides
(Orthene and Diazinon). The statistical
parameters, slope, intercept and cor-
relation coefficients were recorded.
Reproducibility of the method was
tested with two successive runs with
Endothall. The slopes were 143.6 and
136.6 with correlation coefficients of
0.91 and 0.96, respectively. Analysis
of covariance showed there was no
significant difference between these
slopes at the 95% confidence interval.
This Project Summary was devel-
oped by EPA's Environmental Re-
search Laboratory. Corvallis. OR, to
announce key findings of the research
project which is fully documented in a
separate report of the same title (see
Project Report ordering information at
back).
Introduction
A rapid, simple, inexpensive and
reproducible bioassay procedure or
protocol was needed to determine the
deleterious effects of toxic substances
in the environment on vegetation, and
provide a standardized procedure where-
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by laboratories could test these diverse
substances under standard conditions.
To develop this protocol, a decision
was made to use ethylene evolution and
visible injury symptom as indices of
toxicity. Stress-induced ethylene pro-
duction by plants is an indication of an
injury occurring after very mild trauma
or unfavorable growing conditions. The
injury was first observed after very mild
adverse chemical treatment, and was
later caused by insect injury, tempera-
ture extremes, drought, irradiation,
disease, and by wounding, pressure, or
abrasion.
Air pollutants such as ozone and CI2,
which have induced stress ethylene
production in several plants, have been
suggested as a measure of ozone injury
on plants. Some investigators have
advocated the use of ethylene and
ethane production to measure S02
injury.
A spectrum of plant species from
diverse families were to be tested under
standardized conditions with precise
levels of toxic substances and measure-
ment of the amount of stress ethylene
produced. Correlations of dosage and
level of ethylene produced were to be
made using statistical procedures.
Experimental
Two growth chambers which control
temperature, light and humidity were
used for growing the plants.
A gas-chromatograph equipped with
a 2-ml sample loop, and a column of
Poropak N, gave good separation of
ethylene and precise, reproducible
determinations.
Toxicants were applied with pendulum
sprayer; an average deposit of liquid
from 5 applications was 2.69 mg/cm2
with a standard deviation of .06 mg/cm2
(Table 1).
Several different methods were em-
ployed for enclosing treated plants and
sampling the amount of ethylene pro-
duced. The best method consists of
covering the treated plant with a two-
quart widemouth glass jar; a small
aluminum weighing dish is placed in the
bottom of a six-inch saucer. The pot
containing the treated plant is then
placed in the aluminum dish and covered
by the inverted jar. The plant saucer is
filled with water to seal the opening. A
gastight syringe, equipped with a bent
hyprodermic needle, is inserted beneath
the jar to withdraw ethylene samples.
Because incubation temperature
affected ethylene production, causing
Table 1. Reproducibility of Sprayer Delivery
Date 8/22/79 1/24/79 11/29/78 12/27/78
Nozzle # 4OO1 4O01 6501 6501
Distance from Sprayer Tip 30
(cm)
30
45
45
Delivery (Mg/cm2)
\
Mean
Standard Deviation
2.06
2.07
2.14
2.09
2.09
.04
2.63
2.67
2.70
2.66
2.79
2.69
.06
1.18
1.26
1.31
1.26
1.31
1.28
1.31
1.22
1.33
1.31
1.28
.05
1.77
1.77
1.79
1.78
1.76
1.77
1.79
1.68
1.75
1.77
1.76
.03
higher ethylene production at increased
temperatures, the plants were held in a
darkened incubation box and warm air
from the growth chamber held the
temperature at about 24°C for the 24
hours of incubation.
Eight species of plants were tested.
The criteria considered in choosing the
test plants were: fast growth; e.g., rapid
production of leaf tissue, uniformity,
plant habitus; e.g., structural character-
istics that make it suitable for this
particular application, and the potential
for high ethylene production when
exposed to mild stress.
Banana Squash (UCR selection).
Corn (Early Sunglow), Cucumber (Pick-
ling SMR-58), Bush Bean (Blue Lake),
and Kidney Bean (Pink), Radish (Scarlet
Globe), Spinach (Thickleaved Nobel),
and Sunflower (Mammoth 307) were
grown in growth chambers.
Phytar and Endothall, both weed
killers known to cause ethylene produc-
tion, were applied as sprays at low
concentrations. The ethylene accumu-
lated during the 24-hours after this
application was measured (Table 2).
Cucumbers and kidney beans were
selected due to their fast growth and
high ethylene production.
Environmental conditions for growth
and development of the test plants with
kidney beans (pink) and cucumbers
(pickling SMR-58) were to grow them in
six-ounce styrofoam cups (180 ml) with
drainage holes in a commercial product.*
"Jiffy Products of America
250 Town Road
Chicago, IL60185
The seeds were planted two-centi-
meters in cups deep and placed in trays
filled with tap water. When germinated,
the plants were irrigated with one-half
strength NCSU phytotron nutrient solu-
tion. Light intensity in growth chambers
was 322 Einsteins/m'Vsec'1. A 12-
hour photoperiod was compared with a
16-hour photoperiod. Beans grown
under the shorter photoperiod produced
considerable more ethylene after spray-
ing with Endothall and Phytar (Table 3),
but slightly less when treated with
sodium fluoride. The day and night
temperatures and relative humidity
established for beans were 27°C, 21 °C,
and 65%, respectively, and for cucum-
bers 30°C, 26°C, and 80%. Kidney
beans (pink) were ready for testing 9 to
10 days after planting while cucumbers
required 14 days.
A series of tests showed that the
optimum light period following darkness
and prior to spray application for stress
ethylene is between 1.5 hours and 2.5
hours (Table 4). Incubation, in a lighted
growth chamber following spraying and
encapsulation, caused a 11 °C tempera-
ture rise which caused ethylene evolu-
tion in controls; thereafter, the plants
were encapsulated as soon as dry and
incubated in the dark.
To establish some definite period
during which the treated plants were
allowed to evolve ethylene during the
dark incubation, beans were treated
with Endothall and cucumbers with
Phytar. Rates of evolution were recorded
for 24 hours or longer. Both plants had
sigmoid rate curves. The bean plants
had plateaued, or were regressing, at 24
hours. The cucumbers reached a plateai
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between 30 and 46 hours with 70% of
the total having been evolved in 24
hours. Because the beans had plateaued,
and 70% of maximum ethylene had
been produced by the cucumbers, 24
hours was selected as fixed period for
sampling.
Preparation of suitable dilutions of
test compounds which limit water solu-
bility can present problems. Oil-soluble
materials can often be dissolved in
acetone and/or a non-toxic oil, such as
olive oil; and emulsifier added, and a
stable oil-in-water emulsion prepared.
Odorless kerosene can also serve as a
primary solvent. These emulsions can
o'ften be diluted to obtain suitable
concentrations. Less soluble com-
pounds can often be dissolved in acetone
which is then dispersed in water with
violent agitation. Water solutions require
a non-toxic wetting agent to obtain
uniform leaf coverage. In these tests, X-
77* was used.
These procedures were used for
formulation of the compounds tested as
follows:
1. Paraquat - water solution + X-77
2. Phytar - water solution + X-77
3. Endothall - water solution + X-77
4. Sodium Fluoride - water solution +
X-77
5. Sodium Chlorate-water solution +
X-77
6. Orthene - water solution + X-77
7. Diazinon - dissolve in acetone-
dispersed in water + X-77
Stress ethylene produced by plants
when exposed to a toxicant increases
proportionately with the toxicant con-
centration and up to a limit the increases
in the stress-ethylene production can be
modeled using the following equation:
Loge (ethylene concentration) = Loge A +
B (concentration of the toxicant). In this
equation, A is an estimate of the ethylene
production of nontreated plants and B is
the slope parameter which is a measure
of the increase in stress-induced ethyl-
ene production in relation to the stress
concentration. Our studies showed'that
this slope parameter can be used to
express the relative toxicity of aqueous
solution or suspensions of toxicants on
vegetation. All plots and slope parame-
ters are based upon the values on the
linear portion of the curve plus the
control values.
Table 2. Ethylene1 produced by Five Different Plant Species After Spraying with
Phytar or Endothall
9/1
Banana
squash
Corn
Cucumber
Bush
beans
Kidney
beans
PHYTAR
0.0
0.095
0.19
0.38
0.76
1.82
3.04
Age, days
Leaf area, cm2
48
58
86
146
21
190
10
2.8
5.3
11
11
80
—
63
81
144
443
1722
2673
14
67
37
146
669
9
130
45
232
517
744
9
165
ENDOTHALL
0.0
0.01
0.02
0.03
0,04
0,08
0.125
0.250
0.375
0.500
Age, days
Leaf area, cm2
47
74
161
667
17
115
0.5
1.2
1.6
8
—
60
84
151
14
72
16
810
1223
9
130
24
938
2835
9
165
•Colloidal Products Corp.
P.O Box 666
Sausalito, CA 94965
'Parts per billion.
26-8 plants per concentration.
The reproducibility of the method was
determined by two tests with Endothall.
The slopes were, respectively, 143.6
and 136.6; the correlation coefficients
were 0,91 and 0.96. Analysis of covari-
ance showed that there is no significant
difference between these slopes; the
95% confidence interval of the mean is
140 ± 8.0. This interval was determined
for n=40; namely, five concentrations at
eight plants each. For n=30, the interval
would be 140 ± 10 and for n=20, 140 ±
12. These results suggest that a smaller
number of plants per concentration
would not affect the results dramatically.
The relative toxicity of test compounds
was determined as above, and the
slope, intercept and correlation coeffi-
cients, are shown in Table 5.
Evaluation of the seven test com-
pounds (Table 5} shows that correlation
coefficients on some earlier runs showed
little significance. However, as the
relative importance of key experimental
factors was recognized and better con-
trolled, better results were obtained and
reproducibility from run-to-run was
good.
Table 3.
98
98
97
97
99
99
101
101
Ethylene Evolution of Kid-
ney Beans and Cucumbers
Grown with 2 Photoperiods
Endothall (Beans)
12 19 604 3195
16 11 196 774
12 6 609 1833
16 5 133 77
12 16 1363 3229
16 17 326 1437
Phytar (Beans/
0 .30g/l .60g/l
12
16
12
10
171
16
318
256
Run 3 Light Control .020g/l .040g/l
hrs ppb ppb ppb
NaF (Cucumbers)
2.62g/l 5.25 g/l
100 12 19 62 139
100 16 33 51 296
t, US GOVERNMENT PRINTING OFFICE 1*1-757-012/7104
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Table 4. Effect of Light Period before Spray Application on Ethylene Evolution
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