United States
Environmental Protection
Agency
Environmental Monitoring
Systems Laboratory
Las Vegas NV 89114
Research and Development
EPA/600/S4-87/012 June 1987
SEPA Project Summary
Single Laboratory
Evaluation of Phytotoxicity Test
W. R. Lower, A. F. Yanders, and W. W. Sutton
The phytotoxicity method is a screen-
ing test used to predict the potential
impact of chemicals on seed germina-
tion and early seedling growth. Lethal
chemical concentrations are indicated
at the seed germination stage while the
effects of sublethal concentrations can
be assessed from data on early plant
development. However, the early seed-
ling growth phase is not typically
conducted if a particular sample causes
a dramatic inhibition in seed germina-
tion. For testing purposes, such a result
would be sufficient to indicate that the
sample was toxic to plants.
An evaluation of the phytotoxicity
procedure was conducted in order to
establish the data quality that could be
achieved within a single laboratory and
to provide a basis for deciding whether
or not the procedure merits collabor-
ative testing. The tomato plant Lyco-
persicon esculentum was used during
this evaluation, and an insecticide,
sodium pentachlorophenate, was pre-
pared for use as a reference material.
For several steps in the procedure,
minor alterations were shown to def-
initely effect the test result, and the
phytotoxicity protocol was conse-
quently revised to emphasize those
steps where the instructions must be
strictly followed. The single laboratory
precision capability, expressed as a
coefficient of variation, was found to
be 27% for the seed germination phase
and 23% for early seedling growth. A
known test response was established
using 50 jug/g and 5 /ng/g of sodium
pentachlorophenate for seed germina-
tion (EC5o = 65±15) and for early
seedling growth (EC5o = 35 + 10),
respectively.
Limits of reliable measurement for
seed germination were determined to
be 66.5 /jg/g to above 200 /jg/g (for
samples of sodium pentachlorophen-
ate). The only statement that could be
made for early seedling growth was
that the lower limit was determined to
be at or above 10.3 /jg/g and that the
upper limit was not determined. The
seed germination phase was capable of
distinguishing between sample con-
centrations that differed by at least 50
fjg/g. However, the early seedling
growth evaluation did not include
enough concentrations to make an
effective sensitivity determination.
This Project Summary was devel-
oped by EPA's Environmental Monitor-
ing Systems Laboratory, Las Vegas,
NV, to announce key findings of the
research project that is fully docu-
mented in a six separate volumes of the
same title (see Project Report ordering
information at bach).
Introduction
The phytotoxicity test was single
laboratory tested in order to establish the
data quality that could be achieved within
a single laboratory The single laboratory
evaluation provides a basis for deciding
whether or not the method merits
collaborative testing, and it more clearly
defines the procedure's potential for
inclusion as part of an operational
monitoring network. Phases of the single
laboratory test typically include the
identification of procedural variables that
must be carefully controlled (ruggedness
testing), evaluation of method sensitivity,
identification of the limits of reliable
measurement, evaluation of systematic
error (bias), and identification of method
precision and accuracy.
The phytotoxicity method is a screen-
ing test used to predict the potential
-------
impact of chemicals on seed germination
and early seedling growth. Lethal chem-
ical concentrations are indicated at the
seed germination stage while the effects
of sublethal concentrations can be
assessed from data on root and shoot
development. The test procedure is well
documented and, with various modifica-
tions, has been used as a bioassay
procedure for the last thirty years, e.g.,
to detect effects of herbicides and metals
and to determine salinity tolerance. The
final test result for the seed germination
phase is presented as the calculated
concentration (dilution) of sample mate-
rial that will cause a 50 percent reduction
in seed germination. A lethal response
at the seed germination stage typically
means that the early seedling growth
phase need not be conducted. The final
test result for early seedling growth is
also presented as a calculated sample
concentration (dilution) that will cause a
reduction of approximately 50 percent in
seedling growth (i.e., one that results in
an approximate ECso).
Procedural differences have been
noted in some of the previously prepared
phytotoxicity protocols, e.g., the number
of seeds or seedlings required for a valid
test result, the number of different plant
species required for testing, and the
required plant culture conditions
(temperature, light, etc.). Prior to the
current evaluation, the phytotoxicity
protocol was revised to improve the
completeness and clarity of all proced-
ural instructions and was peer reviewed
as a step toward achieving a consensus
procedure. The single laboratory test was
then conducted using the tomato plant
Lycopersicon esculentum (Solanaceae)
and an insecticide, sodium pentachlo-
rophenate, was prepared for use as a
reference material.
Procedure
Phytotoxicity Test
The phytotoxicity protocol tested dur-
ing this evaluation was based on the
1982 procedure prepared by the EPA
Office of Toxic Substances and on the
1978 procedure presented in the Federal
Register for the identification and listing
of hazardous wastes. However, during
the current effort several technical
improvements and modifications were
made to the existing method instructions,
a peer review was also conducted as a
step toward achieving a "consensus"
procedure, and, finally, some revisions
were required based on results of the
ruggedness test. The final protocol also
contains some comparatively non-
specific instructions for (a) assessing
phytotoxic effects of volatile compounds
and for (b) acquiring extracts of solid
material, but these procedures were not
evaluated during the current single
laboratory effort.
The seed germination and early seed-
ling growth phases each progress
through a preliminary and definitive test
sequence, but it is not always necessary
to conduct both tests under routine
operating conditions. A very low or a very
high toxicity result for a respective
preliminary test would constitute a final
test result for the respective phase. In
addition, the early seedling growth phase
would not be conducted at all if a
particular sample caused a 50 percent
or more inhibition to seed germination.
For testing purposes, such a result would
sufficiently indicate that the sample
material is toxic to plants. However, a
very low toxicity, or a no effect test result,
for seed germination does not mean that
the sample would not cause sublethal
toxic effects and, under these conditions,
testing would continue with the early
seedling growth phase.
For both seed germination and early
seedling growth, three different concen-
trations (dilutions) of the sample material
are tested during the preliminary effort,
i.e., 0.01, 1.0, and 100 percent of the
original sample concentration. (Note: The
essentially undiluted sample material,
designated as the 100 percent sample,
is usually somewhat less than 100
percent due to the Hoagland's solution,
solvents, etc.) Based on the results from
the respective preliminary test, four
concentrations are selected for the
definitive effort. The selected concentra-
tions must be chosen from the following
list of seven dilution options, and they
must be four adjacent or sequential
concentrations. The seven dilution
options are 0.01, 0.33, 0.66, 1.0, 33.0,
66.0, and 100 percent of the original
material. No attempt is made to continue
testing at dilutions less concentrated
than 0.01 percent of the original sample.
No additional testing is done to confirm
the validity of the respective ECso esti-
mate, and no additional tests are used
to more thoroughly characterize a dose
response curve.
As mentioned above, positive controls,
negative controls, and, if applicable, a
solvent control, are routinely used during
the test. Acceptance criteria, associated
with each type of control, must be
achieved before the overall test response
is recorded as a valid result.
Various species of plants can be used
for the phytotoxicity test, including
lettuce, cucumber, tomato, soybean,
cabbage, carrot, oat, corn, perennial rye
grass, onion, and winter rye. The current
evaluation was conducted using the
tomato and it is likely that some addi-
tional testing (precision, sensitivity, etc.)
would be necessary if another plant
species were selected. During the seed
germination phase, the pH adjusted test
sample (or control sample) is first added
to a stainless steel seed germinator.
Filter paper (4 sheets of qualitative
course paper placed together and cut to
a predetermined size) is soaked in the
test material, removed, allowed to drain,
and then reversely oriented (top becomes
the bottom) to minimize any chromato-
graphic effect. The seeds are thinly
spread on a tray so that they can be either
picked up using a vacuum planter or
manually transferred to the wetted filter
paper. Fifty seeds are placed on the filter
paper in three horizontal rows at approx-
imately 18, 36, and 54 mm from the top.
Seeds must be alternately spaced from
one row to the next so that the roots can
grow past seeds in the adjacent row.
The filter paper and seeds are then
placed between two glass plates. Spac-
ers and clamps are added to prevent
damage to the seeds and to hold the glass
in a fixed position. The assembly is then
returned to the seed germinator where
the bottom of the plates and filter paper
are immersed in the test solution to a
depth of 1 cm (Figure 1). The germinators
(positive control, negative control, sol-
vent control, and various test sample
dilutions) are then placed in an incubator
and kept at 25°C, in total darkness, for
96 hours (germination time for tomato).
After 96 hours, the number of germi-
nated seeds is counted. For purposes of
this test, the primary root (radicle) must
be at least 3 mm long in order for the
seed to be counted as having germinated.
The germination results are then com-
piled for groups of seeds that were
exposed to the different sample dilutions
and to the various control samples.
The same plant species must be used
during the early seedling growth phase
as was used for the seed germination
phase. Seedling growth is presented as
an increase in plant biomass (dry weight]
and is based on the difference between
seedling dry weight at time of initial
exposure (initial negative control) and
-------
seedling dry weight at the completion of
exposure 168 hours later. Plants must
reach a predetermined stage of
development before the actual test
begins.
Approximately 70 tomato seeds are
added per germinator and are allowed
to germinate in full strength Hoagland's
solution. With the appearance of the first
true leaf (7 to 10 days for tomato plants),
the number of seedlings is reduced to
50 plants per germinator. There will
usually be seven germinators (Figure 2).
The various groups of seedlings then
receive respective dilutions of the sample
material, the positive control sample, or,
if required, the solvent control sample.
One group of 50 seedlings (initial neg-
ative control) is harvested at the start of
the test, dried, and weighed. The remain-
ing groups are kept in temperature, light,
and humidity regulated growth chambers
for 168 hours. A simulated day/night
cycle of 16 hours light, 24°C, 50 to 70
percent humidity x 8 hours dark, 21 °C,
70 to 80 percent humidity is maintained
throughout the test. Fluorescent and
incandescent lights are used to maintain
proper lighting. After 168 hours, all plant
groups are harvested.
The initial negative control dry weight
is used as an approximation of plant
biomass at the beginning of exposure (for
all seedling groups). The weight differ-
ence between initial and final negative
controls represents the increase in plant
biomass that would occur in the absence
of either the sample material, the positive
control, or, if applicable, the solvent
control.
Sample Material
The phytotoxicity test was conducted
several times using different chemicals
before a compound was selected for the
single laboratory evaluation. These
preliminary determinations not only
allowed for the selection of a suitable test
compound, but they also allowed the
laboratory personnel to become more
familiar with the "consensus" protocol
instructions. Many compounds were
available that would inhibit both the
germination and the early growth of
tomato plants, but not all of these
chemicals were available as reference
materials in suitable quantities for single
laboratory testing. A reference material
is not required for the ruggedness
evaluation but, if available, should be
used for determinations of precision,
method sensitivity, and limits of reliable
1 7/8"
2 7/2"
183 /a"
fl
»„
ill
.«
•J-
f
L_
pa
Filter
Glass
4"
cer ,
oajn»;^'''A4 - - -
1
1
i
1
J
:^ (teflon) f\'
7
313/16"l
Basic Test Container
181/4"
13/4"
Figure 1. Seed germination unit for phytotoxicity test.
Figure 2.
Basic Test Container
Early seed/ing growth unit for phytotoxicity test.
measurement. Reference samples
should definitely be used for determina-
tions of accuracy and systematic error.
An insecticide, sodium pentachloro-
phenate, was eventually selected.
Ampules of the compound, prepared as
a reference material, were obtained from
the Environmental Monitoring and Sup-
port Laboratory, Cincinnati, Ohio(EMSL-
Cincmnati). The reference samples were
subsequently diluted to the required
concentration. Even though the samples
were diluted, the material was eventually
exhausted and, unfortunately, this par-
ticular reference compound was not
being prepared on a continuing basis.
Concluding portions of the phytotoxicity
evaluation were conducted using analyt-
ical grade sodium pentachlorophenate
dissolved in deionized water.
Single Laboratory Evaluation
Prior to beginning the single laboratory
test, the method protocol was peer
reviewed as a step toward achieving a
"consensus" procedure. Throughout the
single laboratory effort, the method
procedure and method requirements
-------
(experimental conditions, reagents,
laboratory equipment, testing sequence,
controls, etc } were strictly followed as
they were written in the protocol. It was
therefore of considerable importance
that these written instructions be tech-
nically correct, complete, and as unam-
biguous as possible
The initial phase of the single labor-
atory test was to identify procedural
variables that must be carefully con-
trolled If a procedure is "rugged" it will
not be susceptible to the inevitable,
modest departures in routine and the
final test result will not be altered by
these minor variations When a final test
result is altered by small procedural
variations, the method protocol must be
revised to emphasize that a specific step
must be followed or, in some instances,
to provide an associated step for quality
control
A single concentration of sample
material was used, and seven variables
were selected for testing. The protocol
variations were studied simultaneously
thus requiring only eight separate anal-
yses Basically, the difference between
the "protocol directed" result and the
"protocol altered" result were compared.
When the ruggedness test was com-
pleted, the remaining phases of the
single laboratory evaluation (precision,
method sensitivity, etc.) were conducted
using the revised method procedure
Ten separate determinations were
conducted for each test phase to deter-
mine the method's capability for preci-
sion (using 50/yg/g and 5/vg/g of sodium
pentachlorophenate respectively for
seed germination and early seedling
growth) Each determination represented
a valid test response as required by the
method protocol. The determinations
were conducted sequentially using
ahquots of the respective sample, i.e., a
valid test result was obtained from one
determination before proceeding to the
next until all 10 of the respective assays
had been completed. The precision
estimate was then expressed as a
coefficient of variation.
To determine the method's single
laboratory capability for accuracy (and for
systematic error), the testing laboratory
would need both a standard reference
material and a known method response
(true response)to this reference material.
The Student t-test would then be used
to determine the significance of the
difference between the observed single
laboratory test result and the known true
value. Similarly, to detect a consistently
4
present systematic error, a series of
reference material dilutions would be
used. However, there was no known test
response for the phytotoxicity procedure
and, consequently, the current evalua-
tion could not determine the method's
capability for accuracy. To provide a
known test response for future efforts,
an average response was established
based on 20 separate determinations,
i.e., 20 runs for seed germination using
50pg/g sodium pentachlorophenate and
20 runs for early seedling growth using
5 /jg/g sodium pentachlorophenate. This
effort required only 10 additional deter-
minations for each phytotoxicity test
phase since the respective 10 responses
from the precision effort could be used
as part of the data needed to obtain this
average response to a reference sample.
For purposes of the single laboratory
test, the method's sensitivity was defined
as the method's capability to detect (or
distinguish between) small changes in
sample concentration, i e., concentration
of analyte. Sodium pentachlorophenate
concentrations were selected that were
greater than, and less than, the concen-
tration used during the method precision
determination. If the procedure distin-
guished between samples at the origi-
nally selected concentration interval, the
concentration interval was reduced by
approximately one-half and the addi-
tional samples were tested. Similarly, if
the procedure could not distinguish
between samples at the initial interval,
the concentration interval was
increased. Ten sequential assays were
conducted using each of the selected
concentrations Under routine operating
conditions, the assay would only be
conducted one time per sample and,
consequently, when tabulating data for
sensitivity, non-overlapping standard
deviations (rather than non-overlapping
standard errors) were used to indicate
whether or not the method could distin-
guish between the different samples.
To determine the method's limits of
reliable measurement, an attempt was
made to verify that the method capabil-
ities for sensitivity and precision did not
deteriorate at the upper and lower
extremes of the detection range No
attempt was made to establish an upper
and lower detection limit. Sodium pen-
tachlorophenate concentrations were
selected at what was thought to be an
approximate upper and lower limit of
detection. Ten analyses were conducted
using each concentration to provide
precision data (expressed as a coefficient
of variation). Two additional concentra-
tions were then selected, one at each
extreme of the estimated response range
in order to assess method sensitivity,
i.e.,, a total of two sample concentrations
were now available at each extreme of
the response range. As before, ten
determinations were made using the
newly selected concentrations, and the
method's ability to distinguish between
the respective concentrations was pre-
sented as non-overlapping standard
deviations.
Results and Discussion
Ruggedness
Table 1 provides a summary of the
seven variable ruggedness approach
which was used during this evaluation.
An individual varied condition was either
slightly above or slightly below a "pro-
tocol directed" condition. As noted in the
table, the "protocol directed" conditions
were designated as A through G, and the
varied conditions were designated as a
through g. The evaluation was concerned
with identifying variations in the test
result due to the specific procedural
differences, i e., A-a, B-b, C-c, D-d, E-e,
F-f, and G-g. Each of the eight determi-
nations consisted of a single assay
conducted using eight respective ali-
quots of a single test material. The assay
results are indicated as s, t, u, v, w, x,
y, and z.
The average ofA = (s + t+u+ v)/4
compared with the average of a = (w +
x + y + z)/4 served as a means of
Table 1. Experimental Design fora Seven
Variable Ruggedness Test*
Determination Assay
Number Variables Result
1
2
3
4
5
6
7
8
A B C D E F G
A B c D e f g
A b C d E f g
A b c d e F G
a B C d e F g
a B c d E f G
a b C D e f G
a b c D E F g
s
t
u
V
w
X
Y
z
*Based on W. J. Youden, 1969. The
Collaborative Test, p. 151-158. In Precision
Measurement and Calibration. H. H Ku,
Editor, U.S Department of Commerce,
National Bureau of Standards, 436 pp
-------
assessing the effect of changing variable
A to a. Since each of the two groups of
four determinations contained the other
six variables, twice at the upper case
level and twice at the lower case level,
the effect of these variables (if present)
tended to cancel out leaving only the
effect of changing variable A to a. The
relative effect of the other variables was
also estimated by examining the follow-
ing averages:
B _ (s + t + w + x) b_ (u + v + y + z)
4 4
„ _ (s + u + w + y) _ (t + v + x + z)
\j ~~ ~ C —
4 4
n = (s + t + y + z) . _ (u + v + w + x)
4 4
E = (s + u + x + z) _ (t + v + w + y)
F_ (s + v + w+z) f _ (t + u + x + y)
4 4
„ _ (s + v + x + y) _ (t + u + w + z)
44
After tabulating the above averages,
the differences between each respective
variable was computed, e.g.,
. =_ (s + t+u + v) . (w + x + y + z)
r\ — 3 — * ~
4 4
If one or two variables were having an
effect on the test result, their individual
differences (directed vs. altered) would
be substantially larger than the group of
differences associated with the other
variables. Most of the modest procedural
alterations should have little effect on the
test result since, with few exceptions, the
variations were only of a magnitude that
might have been made by a qualified
laboratory following the written method
protocol
Since the phytotoxicity test was com-
posed of a seed germination phase and
an early seedling growth phase and si nee
both phases contained a preliminary and
definitive segment, the ruggedness
evaluation was conducted four separate
times in order to provide a thorough
evaluation for each test component.
Seven protocol variables were selected
for each respective component, i e , seed
germination preliminary test, seed
germination definitive test, early see-
dling growth preliminary test, and early
seedling growth definitive test. However,
since the preliminary and definitive tests
were conducted separately for each
phase, the overall effect of a single
variable was more difficult to assess (i e ,
effect on final test result).
The ruggedness test indicated several
critical steps in the protocol that must
be conducted exactly as directed. In the
case of seed germination, a consistent
seed size and a consistent number of
seeds were critical for all treatment and
control groups. The specified amount of
solvent (0 01 percent of volume) was also
critical as was maintaining a consistent
exposure time for the sample dilution
groups (96 hours). For the early seedling
growth phase, it was critical that all
harvested plant material be dried for a
consistent amount of time (at the stated
temperature) and that four sample
dilutions be used for the definitive test
As was noted for seed germination, it
was essential that the protocol directed
exposure time for treatment and control
groups be carefully maintained and that
no more than the protocol directed
amount of solvent be added to the test
sample. Based on the current rugged-
ness evaluation, the phytotoxicfty test
was not considered to be a particularly
rugged technique and, for several of the
instructions, a minor procedural altera-
tion will definitely affect the test result
The phytotoxicity protocol was conse-
quently revised to emphasize that, for the
above mentioned steps, no procedural
latitude is allowed and the instructions
must be strictly followed.
Precision
Table 2 provides the results for pre-
cision, sensitivity, and limits of reliable
measurement. Based on 10 separate
determinations using 50 /ug/g of sodium
pentachlorophenate, the seed germina-
tion phase's single laboratory capability
for precision (expressed as a coefficient
of variation) was 27 percent Based on
10 separate determinations using 5 /ug/
g of sodium pentachlorophenate, the
early seedling growth capability for
precision was 23 percent For both test
phases, there was some indication that
the precision was actually better than
these values suggest In the case of seed
germination, the 50 fjg/g concentration
was subsequently shown to be approach-
ing the procedure's lower limit of reliable
measurement Determinations made
using 100 /ug/g samples resulted in an
improved method precision of 16 per-
rent The 5 /ug/g concentration used to
determine precision for early seedling
growth was definitely at or below the
method's limit of reliable measurement
Subsequent determinations using 1 /jg/
g concentrations revealed a dramatic
deterioration in precision to 1 77 percent
In retrospect, the 5 /jg/g concentration
was obviously a poor selection, and the
precision assessment would probably
have been more valid using a greater
concentration of sodium pentachloro-
phenate.
Accuracy
Since a known test response was not
available to assess the method's single
laboratory capability for accuracy and for
systematic error, the current evaluation
established a known response based on
20 respective determinations for seed
germination and for early seedling
growth When using 50 /ug/g sodium
pentachlorophenate samples, the seed
germination EC5o response was 65 ± 15
and, when using 5 /ug/g samples, the
early seedling growth EC50 response was
35 ± 103. Since the sodium pentach-
lorophenate was not an actual reference
material, these results are not as bene-
ficial as had been originally intended.
However, the documented response to
analytical grade sample material will
provide some basis for comparison when
other laboratories are conducting this
procedure.
Method Sensitivity
Method sensitivity was assessed by
non-overlapping standard deviations as
opposed to non-overlapping standard
errors (Table 2). Concentration intervals
used to evaluate the seed germination
phase were approximate differences of
10, 20, 40, 50, and 1 50 /ug/g Since the
12 5 /ug/g concentration was obviously
below the limit of reliable measurement
and since the 42 fjg/g concentration was
also close to the lower limit, there were
not as many specific concentrations for
comparison as had been originally
intended The seed germination phase
effectively distinguished between 200
fjg/g and 100 /ug/g (interval of 100 /jg/
g), between 200 /ug/g and 83 7 /ug/g
(interval of 11 6 /ug/g), and between 83 7
/ug/g and 42 /ug/g (interval of 42 /ug/g)
However, there was slight overlap
between 100 /ug/g and 50 /ug/g which
suggests that a 42 /ug/g interval could
not be consistently achieved The seed
germination procedure's capability for
sensitivity is therefore presented as 50
/ug/g (using sodium pentachlorophenate
as sample material)
The early seedling growth evaluation
did not include enough concentrations to
5
-------
Table 2. Phytotoxicity Test Results Using Concentrations of Sodium Pentachlorophenate in Distilled Water (Values Given are Final Test
Results for Either Seed Germination or for Early Seedling Growth)
Seed Germination Phase
Sequential
Assay
Number
1
2
3
4
5
6
7
8
9
10
n
mean (ECsd
S.D.
C. V. (%)
S.E.
mean ± S.E.
mean ± S.D.
12.5
V9/9*
nontoxic
nontoxic
nontoxic
nontoxic
nontoxic
nontoxic
nontoxic
nontoxic
nontoxic
nontoxic
10
—
—
—
—
—
—
42.0
tJ9/9
79
170
97
88
95
90
75
97
84
81
10
96
27
28
8.5
105-88
123-69
50.0
V9/g
60
58
91
105
60
53
78
57
b
b
8
70
19
27
6.7
77-63
89-51
66.5
P9/9
58
60
71
72
65
64
55
66
71
75
10
66
7
11
2.2
68-64
73-59
83.7
V9/9
34
34
47
49
41
58
46
43
59
56
10
47
9
19
2.8
50-44
56-38
100.0
V9/9
46
40
50
37
33
54
52
47
50
38
10
45
7
16
2.2
47-43
52-38
200.0
ug/9
32
30
22
33
27
22
24
38
32
30
10
29
5
18
1.6
31-27
34-24
10.3
V9/9
27
16
53
28
40
12
13
39
10
20
10
26
14
55
4.4
30-22
40-12
Early Seedling
Growth Phase
5.0
fjg/9
48
43
33
33
51
38
33
35
21
41
10
38
9
23
2.8
41-35
47-29
1.0
V9/9*
nontoxic
nontoxic
80
nontoxic
nontoxic
94
nontoxic
nontoxic
c
c
8
—
—
—
—
—
—
S.D. = standard deviation.
S.E. = standard error.
C. V. = coefficient of variation.
= Calculated sample dilution that will cause either a 50 percent reduction in seed germination or a 50 percent reduction in seedling
growth.
dilution estimates greater than 100 percent indicate a nontoxic response. A comparison of specific no effect values would be meaningless.
"Test response not considered valid based on agreement criteria for positive control results.
^Determinations 9 and 10 not conducted.
make an effective sensitivity determina-
tion. The 1 ug/g concentration noted in
Table 2 was below the limit of reliable
measurement, and the early seedling
growth procedure was not capable of
distinguishing between concentrations
of 10.3 fjg/g and 5.0 ug/g (i.e., essen-
tially a 5 fjg/g interval). The only state-
ment that can be made about the sen-
sitivity for early seedling growth is that
it is greater than 5 /ug/g.
Limits of Reliable Measurement
The limits of reliable measurement are
typically determined through an assess-
ment of accuracy, precision, and sensi-
tivity at the upper and lower limits of the
detection range. As stated previously, the
method's capability for accuracy could
not be determined and, therefore, the
limits of reliable measurement were
based on an assessment of precision and
sensitivity. For both phases of the
phytotoxicity test, the concentrations
ranged from 200 fjg/g to 1 ug/g {Table
2). These concentration selections
proved to be adequate for an assessment
of the lower limit, but they clearly did
not allowfor a determination of the upper
limit.
For seed germination, the lower limit
was probably 66.5 /ug/g because of the
noted deterioration in the coefficient of
variation between test results for 66.5
ug/g and for 50.0 ug/g (i.e., 11 percent
as opposed to 27 percent). In terms of
sensitivity, a concentration of 66.5 ug/
g could not be distinguished from less
concentrated samples of 50 ug/g and 42
ug/g (i.e., respective concentration
intervals of approximately 17 and 25 ug/
g). However, a sample concentration of
66.5 ug/g could be distinguished from
samples of 83.7 ug/g (i.e., interval of
approximately 17 ug/g). The 12.5 ug/g
concentration was definitely below the
method's lower limit. Limits of reliable
measurement for the seed germination
phase have consequently been pre-
sented as 66.5 ug/g to above 200 ug/
g (for samples of sodium pentachloro-
phenate). In the case of early seedling
growth, there was even less information
available. The 1 ug/g concentration was
clearly below the limits of reliable
measurement, and the procedure did not
distinguish between concentrations of
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10.3 /jg/g and 5.0 //g/g. Consequently,
the only statement that can be made for
early seedling growth is that the lower
limit is at or above 10.3 jt/g/g and that
the upper limit has not been determined.
Conclusions
As a result of this single laboratory
evaluation, the phytotoxicity procedure is
much closer to a collaborative testing
stage than it was previously, especially
if the tomato plant continues to be used
as the test species. It is essential that
a reference material (preferably sodium
pentachlorophenate) be available for use
by the collaborating laboratories and that
the known test response, determined
during the current effort, be verified
using the reference samples. It will also
be necessary to acquire a better estimate
for the procedure's limits of reliable
measurement before a collaborative
study is conducted. All assay data from
the current single laboratory evaluation
and a complete copy of the phytotoxicity
method protocol are included in the
project report.
W. R. Lower and A. F. Yanders are with the University of Missouri. Columbia,
MO 65203.
W. W. Sutton is the EPA Project Officer (see below/.
The complete report, entitled "Single Laboratory Evaluation of Phytotoxicity
Test." (Order No. PB 87-188 694/AS: Cost: $24.95) will be available only
from:
National Technical Information Service
5285 Port Royal Road
Springfield. VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Environmental Monitoring Systems Laboratory
U.S. Environmental Protection Agency
' P.O. Box 15027
Las Vegas. NV89114
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United States Center for Environmental Research
Environmental Protection Information
Agency Cincinnati OH 45268
Official Business
Penalty for Private Use $300
EPA/600/S4-87/012
0000329 PS
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60604
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