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

-------
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

-------
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


                                gI|

                                    "REET
                                             60604

-------