&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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