PB 81—15 7901
Development of a Protocol for Testing
Effects of Toxic Substances on Plants
California Univ.
Riverside
Prepared for
Corvallis Environmental Research Lab., OR
Feb 81
U.S. DEPARTMENT OF COMMERCE
National Technical Information Service
NTIS
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PT» 9 1-1 57O01
EPA 600/3-81-006
February 1981
DEVELOPMENT OF A PROTOCOL FOR TESTING
EFFECTS OF TOXIC SUBSTANCES ON PLANTS
by
C. Ray Thompson, Gerrit Kats,.
Philip Dawson and Denise Doyle
Statewide Air Pollution Research Center
University of California
Riverside,. California 92521
Grant No. R806270
Project Officer
David T. Tingey
Plant Physiologist
Corvallis Environmental Research Laboratory
Office of Research and Development
U. S. Environmental Protection Agency
Corvallis^ Oregon; 97330
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TECHNICAL REPORT DATA
1Please read Instructions on the reverse before completing)
1. REPORT NO. 2..
EPA-600/3-81-006
3. RECIPIENT'S ACCESSION NO.
P^HI 1 r "7 ft n i -
4. TITLE AND SUBTITLE
Development of a Protocol for Testing
Effects of Toxic Substances on Plants
S. Rft*CmT DATS J 7 9 0 1
FEBRUARY 1981 ISSUING DATE.
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
C. Ray Thompson, Gerrit Kats, Philip Dawson,
and Denise Dovle
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION'NAME AND ADDRESS
Statewide Air Pollution Research Center
University of Caolifomia
Riverside, California 92521
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
Contract R806270-
12. SPONSORING AGENCY NAME AND ADORESS-
Environmental Research Laboratory-Corvallis
Office of Research and Development
Environmental Protection Agency
Corvallis. Oregon 97330
13. TYPE OF REPORT ANO PERIOD COVERED
final
14. SPONSORING AGENCY CODE
EPA/600/02
15. SUPPLEMENTARY NOTES
16" ABSTRACT This study was designed to devise a rapid, simple, reproducible bioassay pro-
:edure to determine effects of so-called "toxic substances in the environment" on vege-
:ation and provide a standardized procedure for evaluation and comparison of effects of
liverse compounds. Eight different plant species were grown and evaluated for speed of
5rowth,e.g.,rapid production of leaf tissue, uniformity within the particular cultivar,
>lant habitus,e.g.,structural characteristics that make it suitable for this particular
ipplication,. and the potential, for high ethylene production when exposed to mild stress.
)f the varieties grown in the growth chambers, pink kidney beans and cucumbers were se-
jec-ted. as. moat suitable..
Seven compounds were tested by the procedure devised: two organic herbicides: Paraquat,
and Endothall; three inorganic plant toxicants: Phytar, sodium fluoride and sodium
chlorate; and two insecticides: Orthene and Diasinon. The statistical parameters, slope
intercept and correlation coefficient were recorded. Reproducibility of the method was
tested with two successive runs with Endothall.. The slopes were 143.6 and 136.6 with
correlation coefficient of 0.91 and 0.96 respectively. Analysis of covariance showed
there was no significant difference between these slopes at the 95% confidence interval.
17. KEY WORDS AND DOCUMENT ANALYSIS
a. DESCRIPTORS
b. IDENTIFIERS/OPEN ENDED TERMS
c. cosati Field/Croup
18. OISTRI8UTION STATEMENT
Release to Public
19. SECURITY CLASS (This Report)
Unclassified
21. NO. OF PAGES
20. SECURITY CLASS (This page)
Unclassified
22. PRICE
"PA Form 2220-1 (Rev. 4-77) previous edition is obsolete
»
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DISCLAIMER
This report has been, reviewed by the Corvallis Environmental Research
Laboratory, U.S. Environmental Protection Agency, and approved for publica-
tion. Approval does not signify that the contents necessarily reflect the
views and policies of the U.S. Environmental Protection Agency, nor does
mention of trade names or commercial products constitute endorsement or
recommendation for use.
ii
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FOREWORD
Effective regulatory and enforcement actions by the Environmental Protection
Agency would be virtually impossible without sound scientific data on pollu-
tants and their impact on environmental stability and human health. Respon-
sibility for building this data baser has been assigned to EPA's Office of
Research and Development and its 15 major field installations, one of which
is the Corvallis Environmental Research Laboratory.
The primary mission of the Corvallis Laboratory is research on the effects
of environmental pollutants on terrestrial, freshwater, and marine systems;
the behavior, effects and control of pollutants in lakes and streams, and
the development of predictive models on- the movement of pollutants in the
biosphere.
This report, describes the development of a protocol for testing the effects
of toxic substances on plants and details the kind of equipment required
for foliar application of toxicants, cultural conditions for raising
uniform test plants, environmental parameters for Insuring reproducible
responses which is the evolution of stress ethylene and statistical
evaluation of data to yield results which will provide a relative evalua-
tion of the toxicity of a given substance to plants.
T. A. Murphy
Director
iii
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ABSTRACT
The purpose of this study was to devise a rapid, simple, and reproducible
bioassay procedure to determine effects of so-called "Toxic Substances in the
Environment" oh vegetation and provide a standardized procedure for evaluation
and comparison of effects of diverse compounds.
Eight different plant species were grown and evaluated for speed of growth,
e.g., rapid production of leaf tissue, uniformity within the particular cultivar,
plant habitus, e.g., structural characteristics 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 (Pickling
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. Pink kidney beans and cucumbers were selected as most suitable.
The plants were grown in a growth chamber in small plastic pots in a
commercial potting mix; beans for 9-10 days and cucumbers for 14 days prior to
spraying. A photoperiod of 12 hrs was shown to produce plants which evolved
the most stress ethylene. The plants were sprayed with a modified 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 2.0 hrs.
Thirty minutes after, spraying the plants were encapsulated under one-half gal
glass jars with a water seal and were incubated for 24 hours in a dark chamber
at 24°C._ Preliminary range tests with 4 dosages of test compounds on 8
replicates each were used to establish suitable dosages for final evaluation.
When the range of dosages was found where small increases in levels caused
maximum changes in stress ethylene evolution, five dosages were used with 8
replicates for the final evaluation.
Ethylene samples were removed from the jars with a syringe having a bent
needle and concentrations of ethylene were determined with a calibrated
Aerograph 1520 gas chromatograph. the stress ethylene evolved from plants
was plotted by computer vs the amount of compound applied from the equation:
Loge (ethylene concentration) =* Loge A + B (concentration of the toxicant).
Seven compounds were tested by the above procedure: two organic herbi-
cides: Paraquat and Endothall; three inorganic plant toxicants: Phytar,
sodium fluoride and sodium chlorate; and two insecticides: Orthene and
Diazinon. The statistical parameters, slope, intercept and correlation
coefficient were recorded.
Reproducibility of the method was tested with two successive runs with
Endothall. The slopes were 143.6 and 136..6 with correlation coefficient of
0.91 and 0.96 respectively. Analysis of covariance showed there was no
significant difference between these slopes at the 95% confidence interval.
The protocol as devised was prepared for publication in the Bulletin of
Environmental Contamination and Toxicology.
iv
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This report was submitted in fulfillment of Grant R-806270-01 by the
Statewide Air Pollution Research Center, University of California, Riverside
under the sponsorship of the Corvallis Environmental Research Laboratory of
the Environmental Protection Agency. This report covers the period of
8/15/78 to 8/14/80 and work was completed on 10/6/80.
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TABLE OF CONTENTS
Page
Foreword iii
Abstract iv
Introduction 1
Summary of progress 3
A. Assembly of Equipment 3
1. Growth Chambers 3
2. Gas Chromatograph 3.
3. Method of Spray Application 3
4. Collection of Ethylene 4
B. Test plants 5
1.- Species of Plants Tested 5
2. Growth Conditions 5
3. Determination of Optimum Light Period Before
Application of the Toxicant 7
4. Effect of the Light Period After Spraying on
Rate of Etheylene ProdactioTr 7
5. Determination of Incubation Period 8
6. Formulation of Toxic Substances Prior to Application
to Plants 9
7. Effect of Toxic Substances, Evaluation of Data 10
8. Determination of Reproducibility of Method 11
9. Determination of Relative Toxicity of Test
Compounds 11
A.. Endothall 11
B. Phytar 560 11
C. Paraquat
D. NaF
E. Sodium Chlorate
F. Orthene
G. Diazinon
C. Discussion
D. Literature Cited
Tables 1-6
Figures 1-18
12
13
13
13
14
16
17-22
23-41
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DEVELOPMENT OF A PROTOCOL FOR TESTING EFFECTS OF
TOXIC SUBSTANCES ON PLANTS
Introduction
A rapid, simple, inexpensive and reproducible bioassay procedure was
needed to determine the deleterious effects of so-called "Toxic Substances in
the Environment" on vegetation and provide a standardized procedure whereby
laboratories could test these diverse substances under standard conditions.
This would allow direct comparison of results between laboratories arid provide
information to local, state and federal agencies as to the hazards involved
in the use of these substances. Where necessary, suitable controls and
regulations would then be developed.
To develop this protocol it was decided to use ethylene evolution plus
visible injury symptoms, as indices of toxicity. Stress induced ethylene
production by plants has been shown to be an indication of injury which occurs
following very mild trauma or unfavorable growing conditions. It was first
observed by Williamson (1950) and occurs after very mildly adverse conditions
such as chemical treatment (Cooper et al. 1968), insect injury (Galil 1968),
temperature extremes (Vines et al. 1968), drought (McMicheal et al. 1972),
Y irradiation (Williamson 1950), disease (Vines et al. 1968), mechanical
effects such as wounding (Hall 1951) (Saltveit and Dilley 1978), pressure
(Goeschl et al. 1966), or abrasion (Cooper et al. 1968).
Air pollutants such as ozone and CI2 (Abeles and Abeles 1972; Tingey et al.
1978) have been shown to induce stress ethylene production in several plants
and have-been suggested as a measure of ozone injury on plants (Tingey et al.
1976). Bressan et al. (1978) have also suggested the use of ethylene and
ethane production to measure SO2 injury.
It was proposed that a spectrum of plant species from diverse families,
be tested under standardized conditions with precise levels of toxic substances
and the amount of stress ethylene produced measured. Correlations of dosage
and level of ethylene produced would be made by suitable statistical procedures.
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DEVELOPMENT OF A PROTOCOL FOR TESTING EFFECTS OF TOXIC SUBSTANCES ON PLANTS
I. Summary of Progress
A- Assembly of Equipment
1. Refurbish growth chambers and define growth conditions
Two growth chambers from Controlled Environments, Inc., Model
E15P were rebuilt and made operational. These chambers control temperature,
light and humidity.
2. Gas Chromatograph. An Aerograph 1520 gas chromatograph equipped
with a 2 ml. sample loop and flame ionization detector was used for ethylene
measurement. The. instrument was installed in a controlled temperature room.
A column of Poropak N* gave good separation of ethylene and ethane. After
considerable experimentation.the following flow rates and temperatures were
selected to optimize ethylene measurement: N£ 57 ml/min,, H;? 45 ml/min, O2
300 ml/min, and a column temperature at 75°C, detector at 80°C and injector
at 175°C. A standard tank of 1 ppm ethylene was used for calibration..
3. Method of Spray Application. To assure reproducibility and ease
of operation a pendulum sprayer as described by Day et al. (1963) was modified
for this purpose. It consists of a miniature compressed air sprayer mounted
on a 1.8m pendulum equipped to automatically spray a plant placed beneath
the center of its arc of swing (Figure 1).
Character of the deposit and deposition rate are determined by: pressure
at the nozzle tip, nozzle type and size, height above the leaf surface and the
speed with which the nozzle, passes over the plant. The objective was a generous
wetting of the leaf surface without runoff through manipulation of these
variables. The following conditions gave reproducible results. The pendulum
was released 60 cm from the bottom of the arc of swing as measured at the
nozzle tip. Pressure at the nozzle was 30 psi. Nozzle height above the
leaf surface was 30 cm. The nozzle was a**Tee-Jet flat spray tip no. 4001.
The delivery rate was determined by spraying preweighed paper towels or
filter paper and weighing afterwards. One spray application consisted of
two passes of the pendulum. The results showed an average liquid deposit
from 5 applications to be 2.69 mg/cm^ with a standard deviation of .06 rag/cm^.
See representative data in Table 1.
4. Collection of Ethylene and Sampling Procedure. Several different
methods.of enclosing treated plants and sampling the amount of ethylene pro-
duced were tried. Initially plants were encapsulated by placing them inside
polyethylene or mylar bags, supported by wire frames (Figure 2) but these
materials were permeable to ethylene. . Tests with single film polyethylene
or mylar bags showed aloss of 85.5% and 80.1%, respectively of 800 ppb of
ethylene during 24 hrs while glass jars with a water seal lost 4.5%. Other
*Alltech Associates
343 2nd Street
Los Altos, CA 94022
**Spraying Systems Company
North Avenue at Schmale Road
Wheaton, Illinois 60187
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investigators found a multilayer "Cryovac B-620"** bag to lose about 15%.
The final procedure devised is to cover the treated plant with a 2 qt wide-
mouth glass Mason Jar. This is done by placing a small aluminum weighing
dish in the bottom of a 6" plant 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.(Figure 3). A gas
tight syringe equipped with a bent hypodermic needle is inserted beneath the
jar and is used to withdraw ethylene samples. Solubility of ethylene in
water is 1-2% and is ignored as a systematic error.
Incubation temperature affected ethylene production causing higher
production at increased temperatures. To control this variable a-darkened
incubation box was used. Warm air from the growth chamber held the
temperature at about. 24° C for the 24 hours-of incubation (Table 7) .
B. Test Plants
1. Species of Plants Tested. The criteria considered in choosing
the test plants were: fast, growth, e.g., rapid production of leaf tissue,
uniformity within the particular cultivar, plant habitus, e.g., structural
characteristics 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 (Pickling
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., The latter three had a number of undesirable characteristics of
which the: relative slow growth was the most important. The other cultivars
were tested after the,first normal leaf was fully developed.
Phytar and Endothall, both weed killers and known to cause ethylene
production, were applied as sprays at low concentrations. The ethylene,
accumulated during 24 hours after this application was measured (Table 2).
The results show that the rate of ethylene production differs greatly depend-
ing upon the cultivar and the toxicant. Combining the requirement of fast
growth and high ethylene production, cucumbers as well as the kidney, beans
showed superior qualities.
2. Growth Conditions. Environmental conditions proved to affect
not only growth and development of the test plants, but also the ethylene
response although not all environmental parameters were tested exhaustively.
Kidney beans (pink) and cucumbers (pickling SMR-58) were grown in 6-oz styrofoam
cups (180 ml.) provided with drain holes in the bottom. Initially a peat/
sponge rock mix (2:1 vol:vol), was used, but a comtaercial.product called
Jiffy Mixt gave superior results with regard to. uniformity of germination
and saved preparation time. A single test comparing the rate .of ethylene
evolution of plants grown-in two media, Jiffy Mix and Jiffy Mix plus sponge
rock and sprayed with Phytar showed that beans or cucumbers grown in Jiffy
Mix alone gave a marked improvement in uniformity of the amounts of ethylene
evolved as well as the total amount (Table 3).
isieit 4*
Super Saucers Jiffy Products of America
Childs and Associates 250 Town Road
P.O. Box 807 Chicago, IL. 60185
Mill Valley, CA ¦ 94941
•fck
Cyrovac Division
W. R. Grace and Co.
Duncan, SC 29334
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The seeds were planted 2 cm deep in Jtffy Mix moistened with tap water.
No watering was done on the top until the seeds had completely germinated.
This prevented the seeds from rotting and improved uniformity considerably.
Once germinated, the plants were irrigated with one-half strength NCSU
phytotron nutrient solution (Downs and Bonaminio 1976). Light intensity in
growth chambers was 322 y Einsteins/m~^/sec~^. This was close to the maximum
for these chambers. Although light intensity during growth may well have an
effect on the-rate of ethylene production,after treatment, no tests were done,
to determine the optimum. A 12-hour photoperiod was compared with a 16-hr
photoperiod and did not show a significant increase in growth rates or plant
quality, but beans grown under the shorter photoperiod produced considerably
more ethylene after spraying with Endothall and Phytar (Table 4 and Figure 4),
but slightly less.when treated with sodium flouridei
The day and night temperatures and relative humidity established from
previous horticultural experience for beans were 27°C, 21°C, and 657,,
respectively, and for cucumbers 30°C, 26°C, and 80%. Under these conditions
both plant species grew most rapidly while maintaining the quality required
for handling the plants during spraying and encapsulating. Kidney beans
(pink) were considered ready for testing when the primary leaves had reached
a maximum size which was typically 9-10 days after planting. Cucumbers
were used when the first normal leaves reached maximum size which required
14 days.
3. Determination of Optimum Light Period Before Application of the
Toxicant. Preliminary observations showed that application of the toxicant
in the morning gave more ethylene evolution than in afternoon indicating that
the length of the light period following darkness and prior to spray applica-
tion.. affec.ted.-this-response of bean and cucumber plants greatly. A series
of tests showed that there is an optimum light period for stress ethylene.
This optimum, might.also be affected by the toxicant used and would require
a large number of tests for precise determination. Our tests indicated
that toxicant application between 1.5 and, 2.5 hr.would be an adequate guide-
line. It also has logistic advantages because it provides enough time for
the spray application, drying, and encapsulation during which the plants
are exposed to light. The test plants were grown in Jiffy Mix incubated
in 2 qt jars, and the values, shown in Table 5 represent, mean values for
ethylene evolution from 8-10 plants per treatment.
4. Effect of the light period after spraying on the rate of ethylene
production of bean or cucumber plants. The effect of the light period after
spraying, was studied using bean and cucumber plants treated with Endothall,
Phytar and. sodium fluoride. The Endothall concentrations were .02 g/1,
Phytar 0.37 g/1 and NaF 10.59 g/1 for beans. Ear cucumbers the concentra-
tions of Endothall, Phytar and NaF were .040, 0.37 and 5.25 g/1, respectively.
Eight plants per treatment were used and incubation was 24 hrs in the dark.
The plants were allowed to dry in the growth chamber for one half hour
following the spray application. After exposing the plants to a range of
light periods they were encapsulated and placed in the dark. All control
plants evolved less than 20 ppb ethylene. The results (Table 6) show that
the highest ethylene concentration is produced by paints encapsulated and
placed in the dark after one half hour of preincubation time, which is also
the period required for drying. For cucumbers the optimum light period
is approximately 1 hr after spraying.
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An alternative test was done to determine if exposure to light for
different periods after encapsulation had any effect on the rate of ethylene
evolution. All plants were encapsulated one half hour after spraying in order
to dry. Thereafter one series was placed immediately in the dark for 24 hr
and the other exposed to light in the growth chamber for 2 hr and placed in
the dark for 22 hr thereafter.
The results (Table 7) show that cucumber plants as well as the beans not
treated with a toxicant (control) produce more ethylene if 2 hr of light is
given before the dark incubation. A temperature increase of 11°C in the
container during the light period probably causes enough stress to account
for the ethylene increase. This interference alone would make it less
advisable to apply light after encapsulation.
Beans and cucumbers sprayed with Phytar also showed an increase in
ethylene when exposed to 2 hr of light before dark incubation (Table 7).
When sprayed with sodium fluoride, however, there appears to be a reduction
in both cases. We believe that this provides adequate information to decide
that dark incubation for 24 hr immediately after encapsulation is most
desirable.
5. Determination of Incubation Period. 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 hr or longer. Both plants
had sigmoid rate curves (Figure 19). The bean plants had plateaued or were
regressing at 24 hr. The cucumbers reached a plateau between 30 and 46 hr
and 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 hr was selected as a fixed period for sampling. This time is also very
convenient logistically.
6. Formulation of toxic substances prior to application to plants.
Preparation of suitable dilutions of test compounds which have limited water
solubility can present problems. Oil soluble materials can often be dissolved
in acetone and/or a non-toxic oil, such as olive oil, an emulsifier added, and
a stable oil-in-water emulsion prepared. Odorless kerosene can also serve as
a primary solvent. These emulsions can often be diluted ad lib to obtain
suitable concentrations. Less soluble compounds can often be dissolved in
acetone which is then dispersed in water with violent agitation. Compounds
with adequate water solubility but which dissolve very slowly can be .
suspended in a cloth bag in a tank. This allows the dense solution which
forms at the solid-liquid surface to sink thus renewing the dilute solution,
and aids* in rapidity of solution. Water solutions require a non-toxic wetting
agent to obtain uniform leaf coverage. We have used X-77* at 0.625 ml/1.
These procedures were used for formulation of the compounds tested as
follows:
1. Paraquat - water solution + X-77
Colloidal Products Corp.
P.O. Box 666
Sausalito, CA 94965
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2. Phytar - water solution + X-77
3. Endothall - water solution "
4. Sodium Fluoride - water solution "
5. Sodium Chlorate - water solution "
6. Orthene - water solution "
7. Diazinon - Dissolve in acetone-dispersed in water + X-77
7„ Effects of Toxic. Substances, Evaluation of Data. Tingey et al.
(19.76) found that stress ethylene produced by plants when exposed to ozone
increased proportionately with the ozone concentration and that up to a
limit the increase of the stress ethylene production can be modeled using
the following equation: . ,Loge (ethylene conc.) » Loge A + B (concentration,
of the toxicant). In this equation A is an estimate of the ethylene pro-
duction of nontreated plants and B is the slope parameter which is a measure
of the increase in stress-induced ethylene production in relation to the
stress concentration.. Our studies showed, that this slope parameter can be
used to express the relative toxicity of aqueous, solutions or suspensions
of toxicants on vegetation. All plots and slope parameters are based upon
the values on the linear portion of the curve plus the control values.
During the later phases of our work we found that the Loge ethylene.response
is sigmoid, so in the future the control and possibly some of the very low
levels should be excluded. This also implies the need for a number of
concentrations larger than the 5 we used. The general procedure was first
to determine a. range of¦concentrations below the point where the Loge ethylene
response ceases to be linear. Four concentrations of the toxicant over a
wide range using 8 plants per concentration, repeated 2 or 3 times was
usually sufficient for an approximation. After this range finding test
5 concentrations within this range were applied using 8 plants per concen-
tration and the slope of the linear regression of the Loge ethylene response
determined. TKe compounds used to establish this methodology were: Endothall,
Bhytar, sodium fluoride, sodium chlorate, Orthene and Diazinon.
8. Determination of reproducibility of method. In order to determine
the reproducibility of the method,. 2 tests were carried out with Endothall.
The slopes were respectively 143.6 and 136.6 and the correlation coefficients
.91 and .96 (Figures 5-6). Analysis of covariance (Snedecor and Cochran 1978;
Bennettand Franklin 1963) showed that there is no significant difference
between these slopes and that the 95% confidence interval of the mean is.
140 +8.0. This interval was determined for n=40, namely 5 concentrations
at 8 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 concen-
tration would not affect the results dramatically.
9• Determination of relative toxicity of test compounds.
A.. Endothall
Endothall caused visible injury to plants at low levels so
a narrow range was tested. Kidney beans (Run //103) and cucumbers (Run If35)
were used. The concentrations applied ranged from 0.01 to 0-04 grams/liter,
these concentrations are much lower than would be used for weed control.
Kideny beans proved to be very sensitive to Endothall and produced large
amounts of ethylene as shown in Figure 5. This increase in stress ethylene
was apparent at a dosage of 0.01 grams/liter, but visible injury was not
observed until a dosage of 0.02 grams/liter and higher were applied. Cucumbers
showed only a slight increase in ethylene even when visible damage occurred
(Figure 7).
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B. . Phytar 560
Phytar 560, which is also an herbicide, produced high
levels of stress ethylene in both kidney beans and cucumbers. In both
species there was a high correlation between; dosage of Phytar 560 and
ethylene production. Run 102 tested kidney beans (Figure 8) treated with
0.3 to 0.6 g/1. Ethylene increased at each concentration as did injury.
Cucumbers in Run #21 (Figure 9) showed visible damage at lower concentra-
tions than beans. A range of 0.09 to 0.37 g/1 caused substantial ethylene
increases. We recommended that Phytar 560 be used at the rate of 0.30 g/1
as a positive control compound for both test species.
C. Paraquat
Paraquat was different from other compounds tested because
it needs light to cause injury. As previously stated light is usually
accompanied by increased temperatures, therefore, we preferred to use dark'
incubation. Beans and cucumbers which were incubated in the dark produced
very low levels of ethylene and the plants failed to show visible damage
until they were placed in the light. However, even with light incubation,
Paraquat produced very low levels of ethylene compared with Endothall and
Phytar 560. Both kidney beans and cucumbers were tested with a range of
0.3 to 2.4 g/1 of Paraquat. Kidney beans in Run #77 (Figure 10) showed a
slight increase in ethylene at each dosage with some visible damage at all
dosages. Cucumbers in Run #78 (Figure 11) exposed to the same range showed
only a slight increase in ethylene but 100% damage on all test plants.
D. Sodium Fluoride
Sodium fluoride was chosen to evaluate the effect of an
inorganic compound... In Run #42 (Figure 12) kidney beans were treated with
a range of 5.25 to 42.0 g/1 of sodium fluoride and showed increasing levels
of ethylene and visible injury. Cucumbers proved to be more sensitive as
illustrated in Run #94 (Figure 13). A smaller range of 2.6 g/1 to. 21 g/1
was used., but ethylene, levels peaked at 10.5 g/1 although visible injury
continued to increase.
The major difference found in the behavior of plants treated with NaF
and those treated with organic compounds involved incubation temperature.
With higher temperatures the NaF treated plants produced less ethylene while
the organic treated plants produce more (Table 7).
E. Sodium chlorate
Limited testing was performed with NaClO^ because of the
low correlation between.dosage and ethylene production. In Run #55 (Figure 14)
kidney beans were treated/with a range of 6.65 to 26.61 g/1. High levels
of ethylene were produced but visible injury was high at all dosages.
Cucumbers in Run #56 (Figure 15) produced very low levels of ethylene when
exposed to a range of 1.66 to 13.30 g/1, but showed considerable injury.
When higher dosages were tested inury increased but ethylene remained low.
F. Orthene
Orthene is a widely used, broad spectrum insecticide. It
is not considered to be toxic to plants and indeed none of our test plants
showed any visible injury. We used a range of 3.0 to 24.0 g/1 on both
7
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kidney beans and cucumbers. These concentrations are much higher than would
be used for insect control. Kidney beans in Run #45 (Figure 16) showed a
very slight.increase in ethylene at 12.0 g/1 and higher. The cucumbers
treated in Run #44 (Figure 17) showed no increase in ethylene.
G. Diazinon
A second broad spectrum insecticide was found to be more
toxic to cucumbers than Orthene. This compound was not water soluble at the
concentrations studied. It was dissolved in 50% acetone. Cucumbers were
sprayed with 1.32 to 10.56 g/1 Diazinon in Run #43 (Figure 18). An increase
in ethylene was observed in the.5.28 and 10.56 g/1 groups. Slight visible
injury occurred but only in the 10.56 g/1 group.
C. Discussion
The purpose of this study was the development of a rapid, simple,
reproducible, bioassay procedure for determining the relative effects of
"Toxic Substances in the Environment" on vegetation and provide a protocol
whereby investigators could evaluate the many and diverse substances to be
considered. Accordingly, the major effort has been to evaluate the
relative effects of cultural, environmental and manipulative, factors on
the primary response, the evolution of stress ethylene. Because rapidly
growing plants are so sensitive to practically all of. these factors it was
necessary to evaluate, define and standardize each of the operations, if
teproducible responses were to be obtained. Some of the operations had
to be fixed arbitrarily so that the other factors could be studied.
Selection of the two plant species for use from the eight tested, pink
kidney beans and cucumbers, was made because they grew rapidly, gave uniform
sized plants, had large flat leaves which could be sprayed uniformly and
evolved large amounts of stress ethylene. To grow plants of a uniform size
it was extremely important that the seed all germinate promptly when planted.
This necessitated a soil mix, hence "Jiffy Mix," which gave proper moisture,
aeration and mineral nutrients. Also, the watering, temperature and light
regimen must be rigidly controlled if the same kind of plants be produced
from run to run. This will of necessity require that plants be grown ih a
growth chamber.
The spray application with the pendulum sprayer gave very uniform
dosages, proceeded rapidly and was essentially trouble free. By enclosing
the apparatus in a plastic curtain and exhausting vapors contamination of
the laboratory was avoided. The studies of light periods and temperature
regimen were cursory but sufficed to, produce plants which evolved sufficient
stress ethylene for easy measurement by the gas chromatograph. The gas
chromatograph, once calibrated, was essentially trouble free.
Evaluation of the seven test compounds shows that correlation coefficients
on some earlier runs showed little significance- However, as the above-named
factors were recognized and controlled much better results were obtained and
reproducibility from run to run was good, (see runs .#103 and 104, Figures 5
and 6). The sigmoid nature of the response curve could interfere with the
suggested Loge evaluation of the data unless several (7-9) concentrations of
toxicant are applied, the. shape of the curve determined and those falling
on both ends, where less linearity occurs, could then be discarded.
8
-------
Literature Cited
Abeles, A. L. and F. B. Abeles, 1972. "Biochemical Pathways of Stress Induced
Ethylene," Plant Physiol., 50, 496-498.
Bennett, Carl A. and N. L. Franklin, 1963. "Statistical Analysis in Chemistry
and the Chemical Industry," p. 441-2, John Wiley and Son3, New York, N.Y.
Bressan, Ray A., Lloyd G. Wilson,. Lloyd LeCureaux and Philip Filner, 1978.
"Use of Ethylene and Ethane Emissions to Assay for Injury by SO2,1' Abstract
for Annual Meeting, Plant Physiol.j JS1, 93 (#509).
Cooper, W. C., G. K. Rasmussen, B. J. Rogers, P. C. Reece and W. H. Henry,
1968. "Control of Abscission in Agricultural Crops and its Physiological
Basis," Plant Physiol., 43, 1560-1576.
Day, B. E., L. S. Jordan and R. T. Hendrixson,. 1963. 'A Pendulum Sprayer
for Pot Cultures," Weeds, 1]L,. 174-176.
Downs, R. J. and D. P.. Bonaminio, 1976. "Phytotron Procedural Manual for
Controlled Environment Research at Southeastern Plant Environment Laboratories,"
North Carolina Ag. Exp. Sta. Bull if244, 1-37.
Galil, J.,-1968. "An Ancient Technique for Ripening Sycamore Fruit in
Eastern Mediterranean Countries, " Ecotr. Botany, 22;, 178-190".
Goeschl, J.. D., L. Rappaport and H. K. Pratt, 1966. "Ethylene as a Factor
Regulating the Growth of Pea Epicotyls Subjected to Physical Stress,"
Plant Physiol.41, 877-889.
Hall, W. C., 1951. "Studies on the Origin of Ethylene from Plant Tissues."
Bot. Gazette, 113, 55-65.
McMicheal, B. L., W. R. Jordan and R. D. Powell, 1972. "An Effect of Water
Stress on Ethylene Production in Intact Cotton Petioles," Plant Physiol.,
49, 658-660.
Saltveit, M. E., and D. R. Dilley, 1978. "Rapidly Induced Wound Ethylene from
Excised Segments of Etiolated Pea," Plant Physiol., 61, 675-679.
Snedecor, G. W. and W. G. Cochran, 1978. "Statistical Methods," p. 449,
Iowa State Press, Ames, Iowa.
Tingey, D. T., P. Nelson and L. Baird, 1978. "Effect of CI2 on Stress Ethylene
Production," Env. and Experimental Botany, Vol. 00, pp. 00-00, Pergamon Press.
Tingey, D. T., C. Standley and R. W. Field, 1976. "Stress Ethylene Evolution:
A Measure of Ozone Effects on Plants," Atmos. Environ., 10, 969-974.
9
-------
Vines, H. M., W. Grierson and G. J. Edwards, 1968. "Respiration, Internal
Atmosphere and Ethylene Evolution of Citrus Fruit," Proc. Am. Soc. Hort.
Sci., 92, 227-234.
Williamson, C. E., 1950. "Ethylene, a Metabolic Product of Diseased or
Injured Plants," Phytopathology 40, 205-208.
10
-------
Table 1
Reproducibility of Sprayer Delivery
Date 8/22/79 1/24/79 11/29/78 12/27/78
Nozzle if 4001 4001 6501 6501
Distance from Sprayer Tip 30 30 45 45
(cm)
Delivery (Mg/cm2) 2.06 2.63 1.18 1.77
2.07 2.67 1.26 1.77
2.14 2.70 1.31. 1.7.9
2.09 2.66 1.26 1.78
2.79 1.31 1.76
1.28 1.77
1.31 1.79
1.22 1.68
1.33 1.75
1.3-1 1.77
Mean 2.09 2.69 1.28 1.76
Standard Deviation .04 .06 .05 .03
11
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Table 2
Ethylene"*- produced by £ive different plant species after spraying with
PHYTAR or ENDOTHALL
Banana
Bush
Kidney
g/1
squash
Corn
Cucumber
beans
beans
PHYTAR
0.0
48
10
63
37
45
0.095
2.8
81
0.19
5.3
144
0.38
58
11
443
232
0.-76
86
11
1722
146
517
1.82
146
2673
744
3.04
669
Age, days
21
80
14
9
9
Leaf area, cm
190
—
57
130
165
ENDOTHALL
0.0
47
0.5
60
16
24
0.01
74
84
0.02
810
938
0.03
151
0.04
161
1223
2835
0.08
667
0.125
0.250
1.2
0.375
0.500
1.6
Age, days
17
8
14
9
9
2
Leaf area, cm
115
—
72
130
165
^"Parts per billion.
2"
6-8 plants per concentration
12
-------
Table 3
ETHYLENE ACCUMULATED DURING 24 HRS AFTER SPRAYING WITH
PHYTAR AT .37 g/1 USING TWO DIFFERENT SOIL MEDIA
Plant No. Jiffy Mix Jiffy Mix/Sponge Rock (1:2)
1 428 ppb 68 ppb
2 465 186
3 577 279
4 291 50.
5 471 415
6 291 68
7 223 12
8 310 25
Average 382 138
13
-------
Table 4. Ethylene Evolution of Kidney Beans and "Cucumbers Grown with
2 Photoperiods
Endothall (Beans)
Run 3 Light Control .020 g/1 .040 g/i
' hrs ppb ppb ppb
98 12 19 604 3195
98 16 11 196 774
97 12 6- 609 1833
97 16 5 133 77
99 12 16 1363 3229
99 16 17 326 1437
Phytar (Beans)
0 .30 g/1 .60 g/1
101 12 12 171 318
101 16 10 16 256
NaF (Cucumbers)
2.62 g/1 5.25 g/1
100 12 19 62 139
100 16 33 51 296
14
-------
Table 5
EFFECT OF LIGHT PERIOD BEFORE SPRAY APPLICATION
ON ETHYLENE EVOLUTION (ppb)
Beans
Cucumbers
Light Period Endothal .02 r/1 Sodium Fluoride 5.25 g/1
(hrs) Run 58 Run 69 Run 70 Run 51 Run 63 Run 72
0 44.97 (6.4) 0.42 (0) 80.14 (19.7)
0.5 374.54 (5.6) 786.63 (8.6) 77.22 (9.6)
1 20.20 (.6)
1.5 7272.26/ (4.4) 509.81 (27.8) /814.96/ (1.1) /158.40/ (20.2) 68.43 (12.1)
2 /86.4S/ (0)
2.5 7609.71/ (10.7) 523.70 (0) 793.19/ (12.8)
3 27.69 (1.7)
3.5 242.46 (2.4) 489.99 (14.5) 419.53 (0) 21.00 (8.4) 55.51 (4.8)
5.5 155.16 (0) 21.58 (1.2)
7.5 15.73 (0) 3.63 (0)
7 / = maximum response
( ) = nontreated controls
-------
TABLE 6
Effect of Preincubation —^
Period of Ethylene Evolution
BEANS
Preincubation Endothall Phytar NaF
period (hrs)
Run 74
Run 84 Run 85
CUCUMBERS
NaF Phytar Endothall
Run 82 Run 83 Run 87
0-5
1-0
179
53.1
478
382
479
257
66.6 168 62.6
166 291 70
1.5
2.0
2.5
13.1
0.88
0.88
427
237
125
40
219
200
46
—^ Preincubation is the period after spraying and before encapsulation.
Plants were placed in lighted growth chamber, immediately after spraying.
16
-------
Table 7- Ethylene Response of Plants Incubated Under Two Different Conditions
KIDNEY BEANS
Incubation Conditions
24 hrs in dark—^
2 hrs in light—^
22 hrs in dark
PPB Increase
°L Increase over
non-preincuba ted
Run 84
Run 85
Control
18.6
61.2
42.6
2297.
•37 g/1
Phytar
478.1
575.8
97.6
207.
Control
10.6
43.4
32.7
3077.
10.Sg/l
NaF
479
375
-104
-21%
Z4 hrs in dark—'1
1/
2J
2 hrs in light—'
22 hrs in dark
PPB Increase
CUCUMBERS
Run 83
Run 82
.37 g/1 5.25g/1
Control Phytar. Control NaF
11,8 291. y
44.1 603.2
32.2 311.8
7.7 16.6.9
26.8 66.6
19.0 -100.3
Run 87
.04 g/1
Control Endoehall
11.0.
LP-6
37.8 98.8
26.7 28.1
7. Increase over
non-preincubated
2737.
1077.
2497.
-607,
242%
397.
— Plants were allowed to dry in lighted.growth:chamber before encapsulation.
2/
Plants were encapsulated in jars and therefore temperatures rose at least
11°C during that period due to radiation. Chamber temperature 24°C.
17
-------
PARTS DESCRIPTION FOR FIGURE 1
1. Pillow blocks with 1/2" bearing.
2. 1/2" shaft.
3. Teflon washers.
4. Flange with set screw (Lab frame foot).
5. Aluminum cam. 2" x 4 1/2: attached to flange to activate
microswitch (6)• Cam is vertically adjustable through
slotted holes.
6. Microswitch.
7. 1/4" galvanized TEE bored out to accommodate 1/2: shaft.
A hole is drilled and tapped for a. setscrew to fix TEE
to the.shaft.
8. 1/4" nipple.
9. Galvanized reducer 1/2: x 1/4:.
10. 1/2" nipple.
11. Galvanized TEE 1/2" x 1/2: x 1/4". This allows wiring
and pressure tube (15 and 17) inside the pendulum.
12. 1/2"' galvanized pipe.
13- Conduit fitting 1/2" (type C).
14. Main switch (toggle type).
15. Electrical wiring for the microswitch, solenoid (20),
main switch circuit.
16. 3-way valve. One position allows the reservoir to be
filled with the spray formulation and the other the
air: to pressurize, the 'system.
17. 1/4" polyethylene tubing for compressed air supply.
18.. Stainless steel ball joints (18 mm) modified to fit
the; valve and solenoid.
19.. Glass; reservoir; 40 mm diameter,. 220 mm long (ball
joints; included).
Reservoir is normally exposed' to 30 lbs pressure.
Ball, joint size is 18 mm and the clamps (not in
sketch) are the screw, lock type.
20. Solenoid valve with stainless steel valve body.
110-115 volt; 100-psi; 1/8" orifice; 10 watt;
valve no V52 DA 2100 Code no VC7.
Skinner Electric Valve Div.
New Britain, Conn. USA
21. L/4 TT Tee-jet stainless steel spray nozzle
assembly with interchangeable tip. The tip used
in this application is Tee-Jet flat spray tip
no 4001.
Preceding page blank w
-------
Figure 2. Cat-away, side view of bag and £ram encapsulation system
-------
Figure 3. Side view of glass jar encapsulation system.
21
-------
3200
2800
2400
12 hr day
g 1600
1200
800
6 hr day
400
0.02
Endothal, g I"1
0.04
Figure 4
22
-------
1 h
0.0!
Kidney Beans
Slope 143.6
Intercept 2.58
Correlation 0.91
Endothal, g
1-1
Figure 5
23
- 5000
-2000
-1000
500
200 _
c
100
50
20
10
5
1
0.02 0.03 0.04 0.05
CD
C
>%
sz
LjJ
-------
5000
2000
1000
500
200 __
100
Kidney Beans
Slope 136.6
Intercept 3.01
Correlation 0.957
0.01
0
0.02
0.03
0.04
0.05
Endothal, g I"1
Figure 6
24
-------
CD
C
© 3
>v
-C
0
Cucumbers
Slope 19.1
Intercept 4.09
Correlation 0.47
l
l
l
0.005 0.010 0.015 0.020
Endothal, g I"1
Figure 7
- 20
a)
cr.
jD
>1
10 H
til
1
0.025 0.030
25
-------
1000
500
200
5-
100
c
CD
C
>•»
20
LjJ
Kidney Beans
8.42
Slope
Intercept 1.89
Correlation 0.93
0.6
0.4
0.5
0.2
0.3
0.1
Phytar, g f1
Figure 8
26
-------
. 5.5-
C
>%
sz.
¦4—
UJ
100
50
0.42
27
-------
0- 200
100
I
50
c
CD
c
>v
sz
20
Ui
LlI
Kidney Beans
2.02
Slope
Intercept 2.22
Correlation 0.696
06
0.25
0.50 0.75
Paraquat, g f1
1.00
1.25
Figure 10
28
-------
4.8
-100
4.0
50
3.2
20
2.4
1.6
Cucumbers
Slope: 0.137
Intercept 2.64
Correlation 0.14
0.8
2.0
0.5
1.0 1.5
Paraquat, g I"1
2.5
Figure 11
29
-------
1000
- 500
50 ^
CD
C
_0>
20 ^
iLi
10
Pink Kidney Beans
Slope 0.040
Intercept 4.20
Correlation 0.80
- 5
0.
0
14
1
21
28
35
Sodium - Fluoride, g
Figure 12
,-1
42
30
-------
200
100
- 50
c
c
#•
CD
c
0
- 20
CL)
>v.
til
LU
_J
Cucumbers
Slope 0.25
Intercept 2.61
Correlation 0.62
06
1
Sodium Fluoride, g I
Figure. 13
31
-------
1y
oi
0
Kidney Beans
Slope 0.227
Intercept 2.9599
Correlation 0.69
10
15
20
25
Sodium Chlorate, g
,-T
5000
2000
1000
500
200
50
20:
10
5
30
c
0
0)
c
0)
s
Figure 14
32
-------
6
Cucumbers
-200
4
"E
Slope 0.050
Intercept 0.67
Correlation 0.24
-too
- 50
Sodium Chlorate,, g I
-1
20 ®
-5
- 2
Figure 15-
33
-------
Kidney Beans
- 200
4
c
CD
C
3
>%
IjJ
26-
0
o
-o
4
o
o
o
o
o
Slope 0.037
Intercept 0.53
Correlation 0.38
1
8 12
Orthene, g
16
.-1
20
- 100
- 50
¦6 20
- 10
24
Figure 16
34
-------
4.4
80
4.2
4.0
"c
%
ui
3.6
o
o
o
o
o
o
o
-o-
o
o
o
Cucumbers
Slope -0.001
Intercept 3.70
Correlation 0.06
70
60
50 Z
c.
40
Q)
'C
>>
_C
LJ
3.4
o
o
30
3.2
0
8 12
Orthene, g I
16
-1
20
24
Figure 17
35
-------
iLl
200
100
50
20
>s
r"
LlJ
Cucumbers
Slope 0.106
Intercept 3.39
Correlation 0.74
0
8
10
Diazinon, g
,-1
12
Figure 18
36
-------
200
50
1000
u~>
cr
uj 800
CD
2
25
CO
CUCUMBERS, PHY TAR
UJ
100 m
ZD
O
ZD
° 600
UJ
UJ
UJ
' BEANS, ENDOTHAL
_i
^ 400
UJ
H
50
UJ
200
25
48
18 24 30
INCUBATION TIME, HOURS
36
42
Figure 19. Ethylene evolution from cucumbers:treated-with Phytar and beans treated with Endothall.
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