Whole Effluent Toxicity Methods Errata Sheet


United States
Environmental Protectio
^1 M^k. Agency
Office of Water	EPA 821-R-02-012-ES
www.epa.gov	December 2016
Whole Effluent Toxicity Methods
Errata Sheet

-------
EPA has made corrections and clarifications to the following three Whole Effluent Toxicity (WET) methods manuals. These errata were proposed
and promulgated as part of the 2017 Clean Water Act Methods Update Rule for the Analysis of Effluent (82 FR 40836, August 28, 2017; docket
number EPA-HQ-OW-2014-0797).
• Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms, Fifth Edition.
U.S. Environmental Protection Agency, Office of Water, Washington, DC. EPA 821-R-02-012, October 2002. Short title: Acute Freshwater
and Marine Manual.
• Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms, Fourth
Edition. U.S. Environmental Protection Agency, Office of Water, Washington, DC. EPA 821-R-02-013, October 2002. Short title: Chronic
Freshwater Manual.
• Methods for Measuring the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms, Third Edition.
U.S. Environmental Protection Agency, Office of Water, Washington, DC. EPA 821-R-02-014, October 2002. Short title: Chronic Marine
and Estuarine Manual.
Three tables are included in this errata document (one for each method manual, in the above order). Each table specifies:
1. Where in the document the change occurs
2. How the current text reads
3. What correction is being made
4. How the corrected text will read
5. The reason for the correction
WET Methods Errata Sheet
1
Finalized December 2016

-------
Errata for Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms, Fifth Edition. U.S. Environmental
Protection Agency, Office of Water, Washington, DC. EPA 821-R-02-012, October 2002
Text Location
Current Text
Correction Being Made
Corrected Text
Reason For Correction
Section 4, Quality
Assurance, Section
4.4.1, Laboratory Water
Used for Culturing and
Test Dilution Water,
Page 8
Water used for culturing and test
dilution should be analyzed for
toxic metals and organics at least
annually or whenever difficulty is
encountered in meeting minimum
acceptability criteria for control
survival and reproduction or
growth.
Add "test" between
"minimum" and
"acceptability criteria."
Water used for culturing and test dilution should be
analyzed for toxic metals and organics at least annually
or whenever difficulty is encountered in meeting
minimum test acceptability criteria for control survival
and reproduction or growth.
Clarification.
Section 4, Quality
Assurance, Section
4.8.3, Food Quality,
Page 9
For foods (e.g., such as YCT)
which are used to culture and test
organisms, the quality of food
should meet the requirements for
the laboratory water used for
culturing and test dilution water as
described in Section 4.4 above.
Spell out acronym for
YCT. Add "at the
concentration in the
culture or test solutions" at
the end of the sentence.
For foods (e.g., such as Yeast, Cereal leaves, and Trout
chow (YCT)) which are used to culture and test
organisms, the quality of food should meet the
requirements for the laboratory water used for culturing
and test dilution water as described in Section 4.4 above
at the concentration in the culture or test solutions.
Clarification. Define the
YCT components. Analysis
should be at the
concentration in the culture
or in the test solutions so that
the foods meet the
requirements at the feeding
concentrations.
Section 4, Quality
Assurance, Section
4.15.2, Documenting
Ongoing Laboratory
Performance, Page 18
A control chart should be prepared
for each combination of reference
toxicant, test species, test
condition, and endpoint.
Change singular words
"condition" and
"endpoint" to plural.
A control chart should be prepared for each combination
of reference toxicant, test species, test conditions, and
endpoints.
Consistency correction with
Section 4.16.2 in "Chronic
Freshwater Manual" and
"Chronic Marine and
Estuarine Manual."
Section 4, Quality
Assurance, Section
4.15.2, Documenting
Ongoing Laboratory
Performance, Page 18
... for establishing the control
charts. In this technique, a
running plot is maintained for the
toxicity values (X) from
successive tests with a given
reference toxicant (Figure 1), and
endpoints (LC50s) are examined
to determine if they are within the
prescribed limits. The types of
control charts illustrated....
Replace sentence "In this
technique..." with two
sentences - "Successive
toxicity endpoints
(NOECs, IC25s, LC50s,
etc.) should be plotted and
examined to determine if
the results (Xi) are within
prescribed limits (Figure
1). The chart should plot
logarithm of concentration
on the vertical axis against
the date of the test or test
number on the horizontal
axis."
... for establishing the control charts. Successive
toxicity endpoints (NOECs, IC25s, LC50s, etc.) should
be plotted and examined to determine if the results (Xi)
are within prescribed limits (Figure 1). The chart should
plot logarithm of concentration on the vertical axis
against the date of the test or test number on the
horizontal axis. The types of control charts illustrated....
Consistency correction with
Section 4.16.2 in "Chronic
Freshwater Manual" and
"Chronic Marine and
Estuarine Manual."
WET Methods Errata Sheet
2
Finalized December 2016

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Errata for Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms, Fifth Edition. U.S. Environmental
Protection Agency, Office of Water, Washington, DC. EPA 821-R-02-012, October 2002
Text Location
Current Text
Correction Being Made
Corrected Text
Reason For Correction
Section 4, Quality
Assurance, Section
4.15.2, Figure 1,
Documenting Ongoing
Laboratory Performance,
Page 20
upraj»moiiwr(x»s)
Replace the graphs in
Figure 1 with log scale
graphs. Add explanatory
text to Figure 1 legend.
TOXIC IT*1 TEST WITH REFERENCE TOXICANTS
CUMULATIVE TEST NUMBER
Figure 1. Control (cusjis) charts: A, General casei B and C, 48-fi acute tests
with sodium chloride. (B) Fathead minnow (Piniep/iales promelas), and
(C) Cen'odap/im'a Mia, with the individual LCSOs (Triangles),
cumulative LC50 means (dotted line), and upper and lower control
limits of two standard deviations (squares). (Provided by the
Environmental Services Division, U.S. Environmental Protection Agency.
Kansas City, KS).
_Uj»F^COWRjDUlMrr |S + 2S)_
CENTRAL TENDENCY
.LOWER CONTROL UMJTJS, -L2S)_
0 S 10
TOXICITY TEST WITH REFERENCE
CUMUIATIVE TEST
» ~
* ~
Figure 1. Control (cusum) charts: A. General case; B
and C, 48-h acute tests with sodium chloride, (B)
Fathead minnow (Pimephales promelas), and (C)
Ceriodaphnia dubia. Charts are prepared in log format
with individual LC50 data plotted on a graph. Note
Figures B and C are plotted using anti-logarithms. The
mean of the logarithmic LC is the central tendency. The
upper and lower control limits are calculated as the
Correction and clarification.
Cusum charting can either
plot anti-logarithm values for
historical mean and warning
limits on the graph with the
y-axis scaled as logarithm or
plot logarithm values for
historical mean and warning
limits on the graph with the
y-axis as normal arithmetic
scaling. The figures B and C
are revised to match the text
in the manual and are shown
using a log format with the
individual LC or EC data
plotted on each graph.
Cusum charts may be plotted
using antilogarithm values so
that the original units (e.g.,
g/L) are displayed on the
graph. The log values on a
log scaled y-axis or antilog
values on an arithmetic scale
y-axis are not be mixed on a
chart. Note Figures B and C
are redrawn for these graphs
using the antilog values of
the LC50 as many graphics
programs do not allow
enlarging the log scale axis
sufficiently (i.e., expanding
within a log unit), and trends
can be difficult to view;
therefore graphs using
antilogarithm values may be
preferable. Reference:
Environment Canada.
2009. Environment Canada's
Guidance on Using
Logarithms for Reference
Toxicant Warning
Chart. Biological Methods
Section, September.
WET Methods Errata Sheet
3
Finalized December 2016

average LC50 value (± 2S) and plotted on the chart as
horizontal lines serving as visual indicators of any trend
of successive effect endpoints that are divergent results.
Cusum charting can plot logarithm values for historical
mean and warning limits on the graph with the y-axis as
normal arithmetic scaling or plot LC50 values for
historical mean and warning limits on the graph with the
y-axis scaled as logarithm. Antilogarithm values can be
used so that the original units (e.g., g/L) are displayed on
the graph. The log values on a log scaled y-axis or
antilog values on an arithmetic scale y-axis are not be
mixed on a chart. (Data provided by the Environmental
Services Division, U.S. Environmental Protection
Agency, Kansas City, KS).

Section 4, Quality
Assurance, Section
4.16.1, Reference
Toxicants, Page 21
...requiring aquatic toxicity tests.
EMSL-Cincinnati hopes to release
EPA-certified solutions of
cadmium and copper with
accompanying toxicity data for the
recommended test species, for use
as reference toxicants through
cooperative research and
development agreements with
commercial suppliers, and will
continue to develop additional
reference toxicants for future
release. Standard reference
materials....
Delete sentence "EMSL-
Cincinnati hopes to...."
... requiring aquatic toxicity tests. Standard reference
materials....
EMSL-Cincinnati no longer
provides reference toxicants.
Section 5, Facilities and
Equipment, Section 5.4,
Reagents and
Consumable Materials,
Section 5.4.9.4, Dried,
Powdered Leaves
(CEROPHYLL'1. Page
25
Dried, Powdered Leaves
(CEROPHYLL®). Dried,
powdered, cereal leaves (e.g.,
CEROPHYLL® or equivalent) are
available....
Delete term
CEROPHYLL® from
section title. Delete "(e.g.,
CEROPHYLL® or
equivalent)" from first
sentence.
Dried, Powdered Leaves. Dried, powdered, cereal leaves
are available....
CEROPHYLL®, a
commercial product, is no
longer available.
WET Methods Errata Sheet
4
Finalized December 2016

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Errata for Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms, Fifth Edition. U.S. Environmental
Protection Agency, Office of Water, Washington, DC. EPA 821-R-02-012, October 2002
Text Location
Current Text
Correction Being Made
Corrected Text
Reason For Correction
Section 6, Test
Organisms, Section
6.6.4, Transportation to
the Test Site, Page 30
In static tests, marine organisms
can be used at all concentrations
of effluent by adjusting the
salinity of the effluent to a
standard salinity (such as 25%o) or
to the salinity approximating that
of the receiving water, by adding
sufficient dry ocean salts, such as
Forty Fathoms®, or equivalent,
GP2 or hypersaline brine.
Replace "Forty Fathoms®"
with "HW
MARINEMIX®" in
sentence.
.. .by adding sufficient dry ocean salts, such as HW
MARINEMIX®, or equivalent....
FORTY FATHOMS®, a
commercial product, is no
longer available. Equivalent
commercial products, such as
HW MARINEMIX®, are
acceptable replacements.
Section 7, Dilution
Water, Section 7.2.1,
Standard, Synthetic
Dilution Water, Page 31
Standard, synthetic, dilution water
is prepared with deionized water
and reagent grade chemicals or
mineral water (Tables 7 and 8)
and commercial sea salts (FORTY
FATHOMS®, HW
MARINEMIX®) (Table 9).
Delete term "FORTY
FATHOMS®" and add "or
equivalent" to sentence.
...and commercial salts (HW MARINEMIX® or
equivalent) (Table 9).
FORTY FATHOMS®, a
commercial product, is no
longer available. Equivalent
commercial products, such as
HW MARINEMIX®, are
acceptable replacements.
Section 7, Dilution
Water, Section 7.2.4.2,
Standard, Synthetic
Seawater, Page 33
Synthetic seawater can also be
prepared by adding commercial
sea salts, such as FORTY
FATHOMS®, HW
MARINEMIX® or equivalent, to
deionized water. For example,
thirty-one parts per thousand
(31%0) FORTY FATHOMS® can
be prepared by dissolving 31 g of
product per liter of deionized
water. The salinity of the
resulting solutions should be
checked with a refractometer.
Delete "FORTY
FATHOMS®" from the
first sentence. Delete
second sentence.
.. .can also be prepared by adding commercial sea salts,
such as HW MARINEMIX® or equivalent to deionized
water. The salinity of the resulting solutions....
FORTY FATHOMS®, a
commercial product, is no
longer available. Equivalent
commercial products, such as
HW MARINEMIX®, are
acceptable replacements.
Section 7, Dilution
Water, Section7.3.4,
Use of Receiving Water
as Dilution Water, Page
33
Where necessary, the salinity of a
surface water can be increased by
the addition of artificial sea salts,
such as FORTY FATHOMS® or
equivalent, a natural seawater of
higher salinity, or hypersaline
brine.
Replace "Forty Fathoms®"
with "HW
MARINEMIX®" in
sentence.
.. .addition of artificial sea salts, HW MARINEMIX®, or
equivalent....
FORTY FATHOMS®, a
commercial product, is no
longer available. Equivalent
commercial products, such as
HW MARINEMIX®, are
acceptable replacements.
WET Methods Errata Sheet
5
Finalized December 2016

-------
Errata for Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms, Fifth Edition. U.S. Environmental
Protection Agency, Office of Water, Washington, DC. EPA 821-R-02-012, October 2002
Text Location
Current Text
Correction Being Made
Corrected Text
Reason For Correction
Section 9, Acute
Toxicity Test
Procedures, Section
9.5.3, Static Tests, Page
44
Saline dilution water can be
prepared by adding dry salts
(FORTY FATHOMS® or
equivalent, or modified GP2) or
hypersaline brine to de-ionized
water, or a suitable surface
freshwater, to adjust the salinity of
the entire dilution series.
Replace "Forty Fathoms®"
with "HW
MARINEMIX®" and
delete "modified" in
sentence.
.. .adding dry salts (HW MARINEMIX® or equivalent, or
GP2) or....
FORTY FATHOMS®, a
commercial product, is no
longer available. Equivalent
commercial products, such as
HW MARINEMIX®, are
acceptable replacements.
Section 9, Acute
Toxicity Test
Procedures, Section
9.11.1, Feeding, Page 46
The organisms are fed at test
renewal, 48 h after the test is
initiated, if Regional or State
policy requires a 96-h test
duration.
Replace "fed at test
renewal, 48 h after the test
is initiated" with "are
provided food 2 h prior to
test renewal at 48h."
The organisms are provided food 2 h prior to test
solution renewal at 48 h, if Regional or State policy
requires a 96 h test duration.
Clarification of when foods
can be added to match the
text in the tables. The feeding
regimes require that food(s)
be made available while
holding prior to the test; for
some methods (Table 12, 13)
for a minimum of 2 h prior
renewing the test solutions in
a test. Any feeding regimes
require that the food is added
2 h prior to test solution
renewal at 48 h
(recommended); except for
the mysid test, where food is
added daily.
Section 9, Acute
Toxicity Test
Procedures, Section
9.11.2, Feeding, Page 46
Where Artemia nauplii are fed, the
nauplii are first concentrated...."
Add "the <24 hold" after
comma in sentence.
Where Artemia nauplii are fed, the < 24 h old nauplii are
first concentrated ...
Clarification of age of the
brine shrimp to be fed.
Section 9, Acute
Toxicity Test
Procedures, Section
9.171, Summary of Test
Conditions and Test
Acceptability Criteria for
Mysid, Mysidopsis
bahia, Acute Toxicity
Tests With Effluents and
Receiving Waters (Test
Method 2007.0)
(Continued), Table 16,
Line 17, Dilution Water,
Page 60
5-30%o±10%; Uncontaminated
source of seawater, deionized
water mixed with hypersaline
brine or artificial sea salts (HW
MARINEMIX®, FORTY
FATHOMS®, modified GP2, or
equivalent) prepared with MILLI-
Q® or equivalent deionized water
(see Section 7, Dilution Water); or
receiving water (available options)
Delete "FORTY
FATHOMS®," from
sentence.
...or artificial sea salts (HW MARINEMIX®, modified
GP2, or equivalent)....
FORTY FATHOMS®, a
commercial product, is no
longer available. Equivalent
commercial products, such as
HW MARINEMIX®, are
acceptable replacements.
WET Methods Errata Sheet
6
Finalized December 2016

-------
Errata for Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms, Fifth Edition. U.S. Environmental
Protection Agency, Office of Water, Washington, DC. EPA 821-R-02-012, October 2002
Text Location
Current Text
Correction Being Made
Corrected Text
Reason For Correction
Section 11, Acute
Toxicity Data Analysis,
Section 11.2.4.3, Sub-
bullet 6, The Trimmed-
Karber Method, Page 79
A computer program which
estimates the LC50 and associated
95% confidence interval using the
Trimmed-Karber Method, canbe
obtained through the
Environmental Monitoring and
Support Laboratory (EMSL), 26
W. Martin Luther King Drive,
Cincinnati, OH 45268. The
program can be obtained from
EMSL-Cincinnati by sending a
diskette with a written request to
the above address.
Replace ".. .through the
Environmental Monitoring
and Support.. .to the above
address." with
replacement software
information.
... can be obtained by clicking on "contact us" at
htto://www.eDa.eov/med. Anv software capable of
calculating the LC50 and associated 95% confidence
interval using the Trimmed-Karber method canbe used.
Computer program is no
longer available from
Cincinnati. It can be
obtained by sending a request
to EPA using the link. Any
software capable of
completing the calculations is
acceptable.
Section 11, Acute
Toxicity Data Analysis,
Section 11.2.5.3, Sub-
bullet 2, The Probit
Method, Page 81
A machine-readable, compiled,
version of a computer program to
estimate the LCI and LC50 and
associated 95% confidence
intervals using the Probit Method
can be obtained from EMSL-
Cincinnati by sending a diskette
with a written request to the
Environmental Monitoring
Systems Laboratory, 26 W. Martin
Luther King Drive, Cincinnati,
OH 45268.
Replace ".. .from EMSL-
Cincinnati ... Cincinnati,
OH 45268" with
replacement software
information.
... can be obtained by clicking on "contact us" at
htto://www.eDa.eov/med. Anv software capable of
calculating the LCI and LC50 and associated 95%
confidence interval using the Probit method can be used.
Computer program is no
longer available from EMSL-
Cincinnati. It can be obtained
by sending a request to EPA
using the link. Any software
capable of completing the
calculations is acceptable.
Appendix A,
Distribution, Life Cycle,
Taxonomy, and Culture
Methods, Section 3.12.1,
Culture Water, Page 218
Artificial seawater is prepared by
dissolving FORTY-FATHOMS®
or equivalent artificial sea salts in
deionized water to a salinity of 20-
30%o.
Replace "FORTY-
FATHOMS®" with "HW
MARINEMIX®" in
sentence.
.. .prepared by dissolving HW MARINEMIX® or
equivalent artificial sea salts....
FORTY FATHOMS®, a
commercial product, is no
longer available. Equivalent
commercial products, such as
HW MARINEMIX®, are
acceptable replacements.
WET Methods Errata Sheet
8
Finalized December 2016

-------
Errata for Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms, Fourth Edition. U.S. Environmental
Protection Agency, Office of Water, Washington, DC. EPA 821-R-02-013, October 2002
Text Location
Current Text
Correction Being Made
Corrected Text
Reason For Correction
Section 4, Quality
Assurance, Section
4.4.1, Laboratory Water
Used for Culturing and
Test Dilution Water,
Page 10
Water used for culturing and test
dilution should be analyzed for
toxic metals and organics at least
annually or whenever difficulty is
encountered in meeting minimum
acceptability criteria for control
survival and reproduction or
growth.
Add "test" between
"minimum" and
"acceptability criteria."
Water used for culturing and test dilution should be
analyzed for toxic metals and organics at least annually
or whenever difficulty is encountered in meeting
minimum test acceptability criteria for control survival
and reproduction or growth.
Clarification.
Section 4, Quality
Assurance, Section
4.8.3, Food Quality,
Page 11
New batches of food used in
culturing and testing should be
analyzed for toxic organics and
metals or whenever difficulty is
encountered in meeting minimum
acceptability criteria for control
survival and reproduction or
growth.
Add "test" between
"minimum" and
"acceptability criteria."
New batches of food used in culturing and testing should
be analyzed for toxic organics and metals or whenever
difficulty is encountered in meeting minimum test
acceptability criteria for control survival and
reproduction or growth.
Consistency correction with
Section 4.8.3 in "Acute
Freshwater and Marine
Manual" and Section 4.8.3 in
"Chronic Marine and
Estuarine Manual."
Section 4, Quality
Assurance Section 4.16,
Documenting Ongoing
Laboratory Performance,
Page 17
ft
i
n
Ftput 3 Cstt
wwwmiw a
.InM.KU, ,J.|n,rt.r Mini™ -
S 40 19 SO
un>** oomfm. iiMrrflt • i s> g
Replace the graphs in
Figure 1 with log scale
graphs. Add explanatory
text to Figure 1 legend.
J
y
0
a
2
1
UPPER LIMIT (Move the NOEC) A
NOEC VALUES (Xi)
LOWER LIMIT (Below the NOEC!
Correction and clarification.
Cusum charting can either
plot anti-logarithm values for
historical mean and warning
limits on the graph with the
y-axis scaled as logarithm or
plot logarithm values for
historical mean and warning
limits on the graph with the
y-axis as normal arithmetic
scaling. The figures A and B
are revised to match the text
in the manual and are shown
using a log format. Cusum
charts may be plotted using
antilogarithm values so that
the original units (e.g., g/L)
are displayed on the graph.
The log values on a log
scaled y-axis or anti-log
fl 10 18 H
sascrrrTKir wn sawosce Ttra&wrra
teSckasfe 'lA'b-sw&K*:,usbcassib, ^ jcwttSaua; *3X. EC.«ICV
0 5 1$ 20
UPPER CONTROL UMIT (x + 2$) g
CENTRAL TENDENCY
LOWER CONTROL LIMIT (x - 2S)
S 10 15 SB
TOXICITY TEST WITH REFERENCE TOXICANTS
WET Methods Errata Sheet
9
Finalized December 2016

Figure 1. Control (cusum) charts: ForNOEC results
(hypothesis estimates) (A): plot the value of the NOEC
and the values of the adjacent test concentrations of
NOEC as the upper and lower limits. For LC, EC or IC
results (point estimates) (B) plot the actual point
estimate, the cumulative mean (central tendency, middle
line) and the upper and lower control limits (± 2S). The
upper and lower control limits are calculated as the
average IC, EC, LC value (± 2S) and plotted on the
chart as horizontal lines serving as visual indicators of
any trend of successive effect endpoints that are
divergent results. The cusum chart should plot logarithm
of concentration on the vertical axis against the date of
the test or test number on the horizontal axis. Successive
toxicity endpoints (i.e., IC25) should be plotted and
examined to determine if the results are within
prescribed limits. Cusum charts can either plot the data
with the x-axis scaled as logarithms as normal arithmetic
scaling or plot the anti-logarithm values on the graph
with the y-axis scaled as logarithm. The log values on a
log scaled y-axis or antilog values on an arithmetic scale
y-axis are not be mixed on a chart.
values on an arithmetic scale
y-axis are not be mixed on a
chart. Many graphics
programs do not allow
enlarging the log scale axis
sufficiently (i.e., expanding
within a log unit), and trends
can be difficult to view;
therefore graphs using
antilogarithm values may be
preferable. Individual LC,
EC or IC data should be
plotted on a graph. The graph
should present the mean of
the logarithmic LC, EC or
ICs for the central tendency.
The upper and lower control
limits are calculated as the
average LC, EC or IC value
plus-and-minus two standard
deviations and plotted on the
chart as horizontal lines,
serving as visual indicators of
any trend of successive effect
endpoints that are divergent
results. For NOEC toxicity
values, the control limits
would consist of one
concentration interval above
and below the concentration
representing the central
tendency. NOEC values
cannot be averaged (Section
4.14.5); actual values and
adjacent upper and lower
concentrations to the NOEC
value are plotted using the
logarithm procedures
described in Section 4.14.5
and Section 9, Chronic
Toxicity Test Endpoints and
Data Analysis) the NOEC-
LOEC interval is used for the
WET Methods Errata Sheet
10
Finalized December 2016

-------
Errata for Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms, Fourth Edition. U.S. Environmental
Protection Agency, Office of Water, Washington, DC. EPA 821-R-02-013, October 2002
Text Location
Current Text
Correction Being Made
Corrected Text
Reason For Correction




cusum chart. Reference:
Environment Canada.
2009. Environment Canada's
Guidance on Using
Logarithms for Reference
Toxicant Warning
Chart. Biological Methods
Section, September.
Section 11, Fathead
Minnow, Pimephales
promelas, Larval
Survival and Growth
Test Method, Section
11.3.6.1.2, Interferences,
Page 57
Total ammonia concentrations
greater than 5 mg/L in the 100%
effluent are an indicator that
toxicity observed in the test may
be due to ammonia (USEPA,
1992).
Replace "the 100%
effluent are" with "in any
concentration tested may
be...."
Total ammonia concentrations greater than 5 mg/L in any
concentration tested may be an indicator that toxicity
observed in the test may be due to ammonia (USEPA,
1992).
Clarification.
Section 11, Fathead
Minnow, Pimephales
promelas, Larval
Survival and Growth
Test Method 1000.0,
Section 11.10.1.2.3, Test
Procedures, Page 65
Sufficient test solution
(approximately 1500 mL) is
prepared at each effluent
concentration to provide 400 mL
additional volume for chemical
analyses at the high, medium, and
low test concentrations.
Replace "at the high,
medium and low test" with
"for all" test
concentrations.
Sufficient test solution (approximately 1500 mL) is
prepared at each effluent concentration to provide 400
mL additional volume for chemical analysis for all test
concentrations.
Clarification. All solutions
need to have sufficient
volume prepared, not just the
high, medium, and low test
concentrations.
Section 11, Fathead
Minnow, Pimephales
promelas, Larval
Survival and Growth
Test Method 1000.0,
Section 11.10.6.1.2,
Observations During the
Test, Page 67
Temperature and pH are measured
at the end of each 24-h exposure
period....
Insert "beginning and"
between "measured at the"
and "end of each."
Temperature and pH are measured at the beginning and
end of each 24-h exposure period....
Clarification.
Section 11, Fathead
Minnow, Pimephales
promelas, Larval
Survival and Growth
Test Method 1000.0,
Section 11.10.6.1.4,
Observations During the
Test, Page 67
Conductivity, alkalinity, and
hardness are measured in each
new sample (100% effluent or
receiving water) and in the
control.
Delete "(100% effluent or
receiving water)" from
sentence. Insert "before
they are dispensed to the
test chambers" to the end
of the sentence.
Conductivity, alkalinity, and hardness are measured in
each new sample and in the control before they are
dispensed to the test chambers.
Clarification.
WET Methods Errata Sheet
11
Finalized December 2016

-------
Errata for Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms, Fourth Edition. U.S. Environmental
Protection Agency, Office of Water, Washington, DC. EPA 821-R-02-013, October 2002
Text Location
Current Text
Correction Being Made
Corrected Text
Reason For Correction
Section 12, Fathead
Minnow, Pimephales
promelas, Embryo-
Larval Survival and
Teratogenicity Test
Method 1001.0, Section
12.3.5.1.2, Interferences,
Page 113
Total ammonia concentrations
greater than 5 mg/L in the 100%
effluent are an indicator that
toxicity observed in the test may
be due to ammonia (USEPA,
1992).
Replace "the 100%
effluent are" with "in any
concentration tested may
be"....
Total ammonia concentrations greater than 5 mg/L in any
concentration tested may be an indicator that toxicity
observed in the test may be due to ammonia (USEPA,
1992).
Clarification.
Section 12, Fathead
Minnow, Pimephales
promelas, Embryo-
Larval Survival and
Teratogenicity Test
Method 1001.0, Section
12.10.1.2.3, Test
Solutions, Page 117
Sufficient test solution
(approximately 1000 mL) is
prepared at each effluent
concentration to provide 400 mL
additional volume for chemical
analyses.
Change "1000 mL" to
"1500 mL." Add "for all
test concentrations" to end
of sentence.
Sufficient test solution (approximately 1500 mL) is
prepared at each effluent concentration to provide 400
mL additional volume for chemical analyses for all test
concentrations.
Clarification. A volume of
1000 mL is not sufficient for
the test. Consistency
correction with Section
11.10.1.2.3 in "Chronic
Freshwater Manual." All
solutions need to have
sufficient volume prepared,
not just the high, medium and
low test concentrations.
Section 12, Fathead
Minnow, Pimephales
promelas, Embryo-
Larval Survival and
Teratogenicity Test
Method 1001.0, Section
12.10.6.1.2,
Observations During the
Test, Page 119
Temperature and pH are measured
at the end of each 24-h exposure
period in at least one test
chamber....
Add "beginning and"
between "the" and "end."
Temperature and pH are measured at the beginning and
end of each 24-h exposure period in at least one test
chamber....
Clarification.
Section 12, Fathead
Minnow, Pimephales
promelas, Embryo-
Larval Survival and
Teratogenicity Test
Method 1001.0, Section
12.10.6.1.4,
Observations During the
Test, Page 119
Conductivity, alkalinity, and
hardness are measured in each
new sample (100% effluent or
receiving water) and in the
control.
Delete "(100% effluent or
receiving water)". Add
"before they are dispensed
to the test chambers" to
the end of the sentence.
Conductivity, alkalinity, and hardness are measured in
each new sample and in the control before they are
dispensed to the test chambers.
Clarification.
WET Methods Errata Sheet
12
Finalized December 2016

-------
Errata for Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms, Fourth Edition. U.S. Environmental
Protection Agency, Office of Water, Washington, DC. EPA 821-R-02-013, October 2002
Text Location
Current Text
Correction Being Made
Corrected Text
Reason For Correction
Section 13, Daphnid,
Ceriodaphnia dubia,
Survival and
Reproduction Test
Method 1002.0, Section
13.3.6.1.2 Interferences,
Page 142
Total ammonia concentrations
greater than 5 mg/L in the 100%
effluent are an indicator that
toxicity observed in the test may
be due to ammonia (USEPA,
1992).
Replace "the 100%
effluent are" with "in any
concentration tested may
be."
Total ammonia concentrations greater than 5 mg/L in any
concentration tested may be an indicator that toxicity
observed in the test may be due to ammonia (USEPA,
1992).
Clarification.
Section 13, Daphnid,
Ceriodaphnia dubia,
Survival and
Reproduction Test
Method 1002.0, Section
13.10.6.1.2,
Observations During the
Test, Page 157
Temperature and pH are measured
at the end of each 24-h exposure
period in at least one test
chamber....
Add "beginning and"
between "at the" and "of
each 24-h."
Temperature and pH are measured at the beginning and
end of each 24-h exposure period in at least one test
chamber....
Clarification
Section 13, Daphnid,
Ceriodaphnia dubia,
Survival and
Reproduction Test
Method 1002.0, Section
13.10.6.1.4,
Observations During the
Test, Page 157
Conductivity, alkalinity, and
hardness are measured in each
new sample (100% effluent or
receiving water) and in the
control.
Delete "(100% effluent or
receiving water)." Add
"before they are dispensed
to the test chambers" to
end of the sentence.
An aliquot is samples for conductivity, alkalinity, and
hardness in each new sample and in the control before
they are dispensed to the test chambers.
Clarification.
WET Methods Errata Sheet
13
Finalized December 2016

-------
Errata for Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms, Fourth Edition. U.S. Environmental
Protection Agency, Office of Water, Washington, DC. EPA 821-R-02-013, October 2002
Text Location
Current Text
Correction Being Made
Corrected Text
Reason For Correction
Section 13, Daphnid,
Ceriodaphnia dubia,
Survival and
Reproduction Test
Method 1002.0, Section
13.13.3.1, Example of
Analysis of the Daphnid
Reproduction Data, Page
170
The response used in the statistical
analysis is the number of young
produced per adult female, which
is determined by taking the total
number of young produced until
either the time of death of the
adult or the end of the experiment,
whichever comes first. In this
three-brood test, offspring from
fourth or higher broods should not
be counted and should not be
included in the total number of
neonates produced during the test.
An animal that dies before
producing young, if it has not been
identified as a male, would be
included in the analysis with zero
entered as the number of young
produced. The subsequent
calculation of the mean number of
live young produced per adult
female for each toxicant provides
a combined measure of the
toxicant's effect on both mortality
and reproduction.
Add word "live" in the
first (twice), second
(once), and third (twice)
sentences.
The response used in the statistical analysis is the
number of live young produced per adult female, which
is determined by taking the total number of live young
produced until either the time of death of the adult or the
end of the experiment, whichever comes first. In this
three-brood test, offspring from fourth or higher broods
should not be counted and should not be included in the
total number of live neonates produced during the test.
An animal that dies before producing live young, if it has
not been identified as a male, would be included in the
analysis with zero entered as the number of live young
produced. The subsequent calculation of the mean
number of live young produced per adult female for each
toxicant provides a combined measure of the toxicant's
effect on both mortality and reproduction.
Clarification.
Section 14, Green Alga,
Selenastrum
capricornutum, Growth
Test Method 1003.0,
Section 14.10.1.2.3, Test
Procedures, Page 204
Sufficient test solution
(approximately 900 or 1500 mL)
is prepared at each effluent
concentration to provide 400 mL
additional volume for chemical
analyses at the high, medium, and
low test concentrations.
Replace "at the high,
medium, and low" with
"for all test."
Sufficient test solution (approximately 900 or 1500 mL)
is prepared at each effluent concentration to provide 400
mL additional volume for chemical analyses for all test
concentrations.
Clarification. All solutions
need to have sufficient
volume prepared, not just the
high, medium, and low test
concentrations.





Section 14, Green Alga,
Selenastrum
capricornutum, Growth
Test Method 1003.0,
Section 14.10.5.1.2,
Observations During the
Test, Page 206
Temperature and pH are measured
at the end of each 24-h exposure
period in at least one test flask....
Add "beginning and"
before "end."
Temperature and pH are measured at the beginning and
end of each 24-h exposure period in at least one test
flask....
Clarification. The algae test
does not have a daily renewal
of test solution.
WET Methods Errata Sheet
14
Finalized December 2016

-------
Errata for Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms, Fourth Edition. U.S. Environmental
Protection Agency, Office of Water, Washington, DC. EPA 821-R-02-013, October 2002
Text Location
Current Text
Correction Being Made
Corrected Text
Reason For Correction
Appendix C, Dunnett's
Procedure, Section 2.5,
Computer Calculations,
Page 275
...Cincinnati, OH 45268. A
compiled executable version of
the program can be obtained from
EMSL-Cincinnati by sending a
written request to EMSL at 3411
Church Street, Cincinnati, OH
45244.
Replace ".. .EMSL-
Cincinnati. ..." with
replacement software
information.
... can be obtained by clicking on "contact us" at
httD://www.eDa.eov/med. Anv software capable of
calculating an analysis of variance (ANOVA) and a
multiple comparison of treatment means with the control
mean (Dunnett's Procedure) can be used.
Computer program is no
longer available from EMSL-
Cincinnati. It can be obtained
by sending a request to EPA
using the link. Any software
capable of completing the
calculations is acceptable.
Appendix I, Probit
Analysis, 2, Page 309
A compiled, executable version of
the program can be obtained from
EMSL-Cincinnati by sending a
written request to EMSL at 3411
Church Street, Cincinnati, OH
45244.
Replace ".. .EMSL-
Cincinnati ...." with
replacement software
information.
... can be obtained by clicking on "contact us" at
htto://www.eDa.eov/med. Anv software capable of
calculating the EC1 and EC 50 (or LCI and LC50) and
the associated 95% confidence interval using the Probit
method can be used.
Computer program is no
longer available from EMSL-
Cincinnati. It can be obtained
by sending a request to EPA
using the link. Any software
capable of completing the
calculations is acceptable.
Appendix K, Trimmed
Spearman-Karber
Method, Section 10,
Page 318
A computer program which
estimates the LC50 and associated
95% confidence interval using the
Trimmed Spearman-Karber
Method, can be obtained from
EMSL-Cincinnati by sending a
written request to EMSL, 3411
Church Street, Cincinnati, OH
45244.
Replace ".. .EMSL-
Cincinnati ...." with
replacement software
information.
... can be obtained by clicking on "contact us" at
htto://www.eDa.eov/med. Anv software capable of
calculating the LC50 and associated 95% confidence
interval using the Trimmed Spearman-Karber Method
can be used.
Computer program is no
longer available from EMSL-
Cincinnati. It can be obtained
by sending a request to EPA
using the link. Any software
capable of completing the
calculations is acceptable.
Appendix M, Linear
Interpolation Method,
Section 7.1, Page 329
To obtain the program and
supporting documentation, send a
written request to EMSL-
Cincinnati at 3411 Church Street,
Cincinnati, OH 45244.
Replace ".. .EMSL-
Cincinnati. ..." with
replacement software
information.
... can be obtained by clicking on "contact us" at
htto://www.eDa.eov/med. Anv software capable of
calculating the mean and standard deviation of the
bootstrapped ICp and associated 95% confidence
intervals can be used.
Computer program is no
longer available from EMSL-
Cincinnati. It can be obtained
by sending a request to EPA
using the link. Any software
capable of completing the
calculations is acceptable.
WET Methods Errata Sheet
15
Finalized December 2016

-------
Errata for Methods for Measuring the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms, Third Edition.
Protection Agency, Office of Water, Washington DC. EPA 821-R-02-014, October 2002
U.S. Environmental
Text Location
Current Text
Correction Being Made
Corrected Text
Reason For Correction
Section 4, Quality
Assurance, Section 4.16,
Documenting Ongoing
Laboratory Performance,
Page 11
I tmoctrmsM A
i&mooKmium
,i 1 , U,H,,L,uXi MnU.WntnJnn*
3 1 to 11 20
I WWCWflWXLftirfttJW I
1
® ic^coNmiMr(*.23t
¦ LM-inl U-hO,»
ft 9 ta IS 20
TOTQFf TEST
Replace the graphs in
Figure 1 with log scale
graphs. Add explanatory
text to Figure 1 legend.
UPPER LIMIT (Above the NOEQ A
1
| hfQEC VALUES (Xfl
%
s
LOWER LIMIT {Below the NOEC)
0 5 10 15 20
Toxicity Test with Reference Toxicant
uppEncowfiotumns + isj B
0
1
S
C£fJTR»l TENDENCY
1
0
3
j*_-js)
0 » « « m
TOXICITY TUT WITH REFERENCE TOXICA* T5
Figure 1. Control (cusum) charts: For NOEC results
(hypothesis estimates) (A): plot the value of the NOEC
and the values of the adjacent test concentrations of
NOEC as the upper and lower limits. For LC, EC or IC
results (point estimates) (B) plot the actual point
estimate, the cumulative mean (central tendency, middle
line) and the upper and lower control limits (± 2S). The
upper and lower control limits are calculated as the
average IC, EC, LC value (± 2S) and plotted on the
chart as horizontal lines serving as visual indicators of
any trend of successive effect endpoints that are
divergent results. The cusum chart should plot logarithm
of concentration on the vertical axis against the date of
the test or test number on the horizontal axis. Successive
toxicity endpoints (i.e., IC25) should be plotted and
examined to determine if the results are within
prescribed limits. Cusum charts can either plot the data
with the x-axis scaled as logarithms as normal arithmetic
Correction and clarification.
Cusum charting can either
plot anti-logarithm values for
historical mean and warning
limits on the graph with the
y-axis scaled as logarithm or
plot logarithm values for
historical mean and warning
limits on the graph with the
y-axis as normal arithmetic
scaling. The figures A and B
are revised to match the text
in the manual and are shown
using a log format. Cusum
charts may be plotted using
antilogarithm values so that
the original units (e.g., g/L)
are displayed on the graph.
The log values on a log
scaled y-axis or antilog
values on an arithmetic scale
y-axis are not be mixed on a
chart. Many graphics
programs do not allow
enlarging the log scale axis
sufficiently (i.e., expanding
within a log unit), and trends
can be difficult to view;
therefore graphs using
antilogarithm values may be
preferable. Individual LC,
EC or IC data should be
plotted on a graph. The graph
should present the mean of
the logarithmic LC, EC or
ICs for the central tendency.
The upper and lower control
limits are calculated as the
average LC, EC or IC value
plus-and-minus two standard
deviations and plotted on the
chart as horizontal lines,
serving as visual indicators of
WET Methods Errata Sheet
17
Finalized December 2016

scaling or plot the anti-logarithm values on the graph
with the y-axis scaled as logarithm. The log values on a
log scaled y-axis or antilog values on an arithmetic scale
y-axis are not be mixed on a chart.
any trend of successive effect
endpoints that are divergent
results. For NOEC toxicity
values, the control limits
would consist of one
concentration interval above
and below the concentration
representing the central
tendency. NOEC values
cannot be averaged (Section
4.14.5); actual values and
adjacent upper and lower
concentrations to the NOEC
value are plotted using the
logarithm procedures
described in Section 4.14.5
and Section 9, Chronic
Toxicity Test Endpoints and
Data Analysis) the NOEC-
LOEC interval is used for the
cusum chart. Reference:
Environment Canada.
2009. Environment Canada's
Guidance on Using
Logarithms for Reference
Toxicant Warning
Chart. Biological Methods
Section, September.
Section 4, Quality
Assurance, Section
4.17.1, Reference
Toxicants, Page 16
. . .requiring aquatic toxicity tests.
EMSL-Cincinnati hopes to release
EPA-certified solutions of
cadmium and copper for use as
reference toxicants through
cooperative research and
development agreements with
commercial suppliers, and will
continue to develop additional
reference toxicants for future
release. Standard reference
materials....
Delete sentence "EMSL-
Cincinnati hopes to ..."
. . .requiring aquatic toxicity tests. Standard reference
materials....
EMSL-Cincinnati no longer
provides reference toxicants.
WET Methods Errata Sheet
18
Finalized December 2016

-------
Errata for Methods for Measuring the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms, Third Edition. U.S. Environmental
Protection Agency, Office of Water, Washington DC. EPA 821-R-02-014, October 2002
Text Location
Current Text
Correction Being Made
Corrected Text
Reason For Correction
Section 5, Facilities,
Equipment, and
Supplies, Section 5.1,
General Requirements,
Page 19
Water used for rearing, holding,
acclimating, and testing organisms
may be natural seawater or water
made up from hypersaline brine
derived from natural seawater, or
water made up from reagent grade
chemicals (GP2) or commercial
(FORTY FATHOMS® or HW
MARINEMIX®) artificial sea salts
when specifically recommended in
the method.
Delete "FORTY
FATHOMS®" from
sentence.
.. .or water made up from reagent grade chemicals (GP2)
or commercial (HW MARINEMIX®) artificial sea
salts....
FORTY FATHOMS®, a
commercial product, is no
longer available.
Section 6, Test
Organisms, Section
6.6.4, Transportation to
the Test Site, Page 25
.. .by adding sufficient dry ocean
salts, such as FORTY
FATHOMS®, or equivalent, GP2,
or hypersaline brine.
Delete "FORTY
FATHOMS®, or
equivalent," in sentence.
.. .by adding sufficient dry ocean salts, such as GP2, or
hypersaline brine.
FORTY FATHOMS®, a
commercial product, is no
longer available.
Section 7, Dilution
Water, Section 7.2.1,
Types of Dilution, Page
26
Standard, synthetic, dilution water
is prepared with deionized water
and reagent grade chemicals (GP2)
or commercial sea salts (FORTY
FATHOMS®, HW
MARINEMIX®) (Table 3).
Delete "FORTY
FATHOMS®," and add "or
equivalent" after "HW
MARINEMIX®" in
sentence.
...or commercial artificial sea salts (HW MARINE
MIX® or equivalent) (Table 3).
FORTY FATHOMS®, a
commercial product, is no
longer available. Equivalent
commercial products, such as
HW MARINEMIX®, are
acceptable replacements.
Section 7, Dilution
Water, Section7.2.3.2,
Standard Synthetic Sea
Water, Page 27
Synthetic seawater can also be
prepared by adding commercial sea
salts, such as FORTY
FATHOMS®, HW
MARINEMIX®, or equivalent, to
deionized water. For example,
thirty-one parts per thousand
(31%0) FORTY FATHOMS® can
be prepared by dissolving 31 g of
sea salts per liter of deionized
water. The source water for the
deionizer can be ground water or
tap water.
Delete "FORTY
FATHOMS®" from first
sentence. Delete second
sentence.
Synthetic seawater can be prepared by adding
commercial sea salts, such as HW MARINEMIX®, or
equivalent, to deionized water. The source water for the
deionizer....
FORTY FATHOMS®, a
commercial product, is no
longer available. Equivalent
commercial products, such as
HW MARINEMIX®, are
acceptable replacements.
WET Methods Errata Sheet
19
Finalized December 2016

-------
Errata for Methods for Measuring the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms, Third Edition. U.S. Environmental
Protection Agency, Office of Water, Washington DC. EPA 821-R-02-014, October 2002
Text Location
Current Text
Correction Being Made
Corrected Text
Reason For Correction
Section 7, Dilution
Water, Section7.3.3,
Use of Receiving Water
as Dilution Water, Page
28
Where necessary, the salinity of a
surface water can be increased by
the addition of artificial sea salts,
such as FORTY FATHOMS®, HW
MARINEMIX®, or equivalent,
GP2, a natural seawater of higher
salinity, or hypersaline brine.
Delete "FORTY
FATHOMS®," from
sentence.
... addition of artificial sea salts, such as HW
MARINEMIX®, or equivalent, GP2, a natural seawater....
FORTY FATHOMS®, a
commercial product, is no
longer available. Equivalent
commercial products, such as
HW MARINEMIX®, are
acceptable replacements.
Section 11, Sheepshead
Minnow, Cyprinodon
variegatus, Larval
Survival and Growth
Test Method 1004.0,
Section 11.3.6.1.2,
Interferences, Page 56
Total ammonia concentrations
greater than 5 mg/L in the 100%
effluent are an indicator that
toxicity observed in the test may
be due to ammonia (USEPA,
1992).
Replace "the 100%
effluent are" with "any
concentration tested may
be."
Total ammonia concentrations greater than 5 mg/L in any
concentration tested may be an indicator that toxicity
observed in the test may be due to ammonia (USEPA,
1992).
Clarification.
Section 11, Sheepshead
Minnow, Cyprinodon
variegatus, Larval
Survival and Growth
Test Method 1004.0,
Section 11.10.1.3.1,
Dilution Water, Page 68
Dilution water may be
uncontaminated natural seawater
(receiving water), HSB prepared
from natural seawater, or artificial
seawater prepared from FORTY
FATHOMS® or GP2 sea salts (see
Table 1 and Section 7, Dilution
Water).
Replace "FORTY
FATHOMS®" with "HW
MARINEMIX®, or
equivalent," and replace
"1" with "3" in sentence.
... or artificial seawater prepared from HW
MARINEMIX®, or equivalent, or GP2 sea salts (see
Table 3, Section7....
FORTY FATHOMS®, a
commercial product, is no
longer available. Equivalent
commercial products, such as
HW MARINEMIX®, are
acceptable replacements.
Consistency with Section 11,
Table 3, Summary of Test
Conditions..., Line 18,
Dilution Water, Page 79
Section 11, Sheepshead
Minnow, Cyprinodon
variegatus, Larval
Survival and Growth
Test Method 1004.0,
Section 11.10.1.2.3,
Effluents, Table 2,
Footnote 1, Page 69
The salinity of the initial 6,800
mL of 100% effluent is adjusted to
20%oby adding 136 g of dry
artificial sea salts (FORTY
FATHOMS®).
Replace "136 g of dry
artificial sea salts (FORTY
FATHOMS®)" with "an
appropriate amount of dry
artificial sea salts (e.g.,
HW MARINEMIX®)" in
sentence.
.. .by adding an appropriate amount of dry artificial sea
salts (e.g., HW MARINEMIX®).
FORTY FATHOMS®, a
commercial product, is no
longer available. Equivalent
commercial products, such as
HW MARINEMIX®, are
acceptable replacements.
Section 11, Sheepshead
Minnow, Cyprinodon
variegatus, Larval
Survival and Growth
Test Method 1004.0,
Section 11.10.7.1.2,
Observations During the
Test, Page 71
Temperature, pH, and salinity are
measured at the end of each 24-h
exposure period in one test
chamber at each test concentration
and in the control.
Add "beginning and"
between "at the" and "end
of." Add "at least one"
between "period in" and
"test chamber."
Temperature, pH, and salinity are measured at the
beginning and end of each 24-hr exposure period in at
least one test chamber at each test concentration and in
the control.
Clarification.
WET Methods Errata Sheet
20
Finalized December 2016

-------
Errata for Methods for Measuring the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms, Third Edition. U.S. Environmental
Protection Agency, Office of Water, Washington DC. EPA 821-R-02-014, October 2002
Text Location
Current Text
Correction Being Made
Corrected Text
Reason For Correction
Section 11, Sheepshead
Minnow, Cyprinodon
variegatus, Larval
Survival and Growth
Test Method 1004.0,
Section 11.11, Summary
of Test Conditions and
Test Acceptability
Criteria, Table 3, Line
18, Page 79
Uncontaminated source of natural
seawater; deionized water mixed
with hypersaline brine or artificial
sea salts (HW MARINEMIX®,
FORTY FATHOMS®, GP2 or
equivalent) (available options)
Delete "FORTY
FATHOMS®," from
sentence.
...or artificial sea salts (HW MARINEMIX®, GP2 or
equivalent)....
FORTY FATHOMS®, a
commercial product, is no
longer available. Equivalent
commercial products, such as
HW MARINEMIX®, are
acceptable replacements.
Section 12, Sheepshead
Minnow, Cyprinodon
variegatus, Embryo
Larval Survival and
Tertatogenicity Test
Method 1005.0, Section
12.6.10.3.8, Reagents
and Consumable
Materials, Table 1,
Footnote 1, Page 126
The salinity of the initial 4000 mL
of 100% effluent is adjusted to
20%o by adding 80 g of dry
artificial sea salts (HW
MARINEMIX or FORTY
FATHOMS®), and mixing for 1 h.
Delete "FORTY
FATHOMS®," from
sentence, add "®" after
"MARINEMIX", and add
"equivalent" after "or" in
sentence.
...dry artificial sea salts (HW MARINEMIX® or
equivalent) and mixing for 1 h.
FORTY FATHOMS®, a
commercial product, is no
longer available. Equivalent
commercial products, such as
HW MARINEMIX®, are
acceptable replacements.
Section 12, Sheepshead
Minnow, Cyprinodon
variegatus, Embryo
Larval Survival and
Tertatogenicity Test
Method 1005.0, Section
12.10.1.3.1, Dilution
Water, Page 131
Dilution water may be
uncontaminated natural seawater
(receiving water), HSB prepared
from natural seawater, or artificial
seawater prepared from FORTY
FATHOMS® or GP2 sea salts (see
Table 3 in Section 7, Dilution
Water).
Replace "FORTY
FATHOMS®" with "HW
MARINEMIX® or
equivalent" in sentence.
... artificial seawater prepared from HW MARINEMIX®
or equivalent or GP2 sea salts....
FORTY FATHOMS®, a
commercial product, is no
longer available. Equivalent
commercial products, such as
HW MARINEMIX®, are
acceptable replacements.
Consistency with Section 12,
Table 2, Summary of Test
Conditions..., Line 16,
Dilution Water, Page 137
Section 12, Sheepshead
Minnow, Cyprinodon
variegatus, Embryo
Larval Survival and
Tertatogenicity Test
Method 1005.0, Section
12.10.6.1.2,
Observations During the
Test, Page 132
Temperature, pH, and salinity are
measured at the end of each 24-h
exposure period in one test
chamber at each test concentration
and in the control.
Add "beginning and"
between "measured at the"
and "end of each." Add
"at least" between
"exposure period in" and
"one test chamber."
Temperature, pH, and salinity are measured at the
beginning and end of each 24-hr exposure period in at
least one test chamber at each test concentration and in
the control.
Clarification.
WET Methods Errata Sheet
21
Finalized December 2016

-------
Errata for Methods for Measuring the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms, Third Edition. U.S. Environmental
Protection Agency, Office of Water, Washington DC. EPA 821-R-02-014, October 2002
Text Location
Current Text
Correction Being Made
Corrected Text
Reason For Correction
Section 12, Sheepshead
Minnow, Cyprinodon
variegatus, Embryo
Larval Survival and
Tertatogenicity Test
Method 1005.0, Section
12.12.1, Summary of
Test Conditions and Test
Acceptability Criteria,
Table 2, Line 16,
Dilution Water, Page
137
Uncontaminated source of natural
seawater; deionized water mixed
with hypersaline brine or artificial
sea salts (HW MARINEMIX®,
FORTY FATHOMS®, GP2, or
equivalent) (available options)
Delete "FORTY
FATHOMS®," from
sentence.
...or artificial sea salts (HW MARINEMIX®, GP2, or
equivalent)....
FORTY FATHOMS®, a
commercial product, is no
longer available. Equivalent
commercial products, such as
HW MARINEMIX®, are
acceptable replacements.
Section 13, Inland
S ilverside, Menidia
beryllina, Larval
Survival And Growth
Method 1006.0, Section
13.10.1.3.1, Dilution
Water, Page 169
Dilution water may be
uncontaminated natural seawater
(receiving water), HSB prepared
from natural seawater, or artificial
seawater prepared from FORTY
FATHOMS® or GP2 sea salts (see
Table 3 in Section 7, Dilution
Water).
Replace "FORTY
FATHOMS®" with "HW
MARINEMIX®" in
sentence.
... or artificial seawater prepared from HW
MARINEMIX® or GP2....
FORTY FATHOMS®, a
commercial product, is no
longer available. Equivalent
commercial products, such as
HW MARINEMIX®, are
acceptable replacements.
Consistency with Section 13,
Table 3, Summary of Test
Conditions..., Line 18,
Dilution Water, Page 179.
Section 13, Inland
S ilverside, Menidia
beryllina, Larval
Survival And Growth
Method 1006.0, Section
13.10.7.1.2,
Observations During the
Test, Page 171
Temperature, pH, and salinity are
measured at the end of each 24-hr
exposure period in one test
chamber at all test concentrations
and in the control.
Add "beginning and"
between "measured at the"
and "end of each." Add
"at least" between
"exposure period in" and
"one test chamber."
Temperature, pH, and salinity are measured at the
beginning and end of each 24-hr exposure period in at
least one test chamber at all test concentrations and the
control.
Clarification.
WET Methods Errata Sheet
22
Finalized December 2016

-------
Errata for Methods for Measuring the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms, Third Edition. U.S. Environmental
Protection Agency, Office of Water, Washington DC. EPA 821-R-02-014, October 2002
Text Location
Current Text
Correction Being Made
Corrected Text
Reason For Correction
Section 14, Mysid,
Mysidopsis bahia,
Survival, Growth, and
Fecundity Test Method
1007.0, Section
14.10.8.1.2,
Observations During the
Test, Page 229
Temperature, pH, and salinity are
measured at the end of each 24-hr
exposure period in one test
chamber at each test concentration
and control.
Add "beginning and"
between "measured at the"
and "end of each." Add
"at least" between
"exposure period in" and
"one test chamber."
Temperature, pH, and salinity are measured at the
beginning and end of each 24-hr exposure period in at
least one test chamber at each test concentration and
control.
Clarification.
Section 14, Mysid,
Mysidopsis bahia,
Survival, Growth, and
Fecundity Test Method
1007.0, Section
14.10.10.3.1,
Termination of the Test,
Page 230
Examine each replicate under a
stereomicroscope (240X) to
determine the number of immature
animals....
Replace "240X" with
"24X."
Examine each replicate under a stereomicroscope (24X)
to determine the number of immature animals....
Typographical error.
Section 14, Mysid,
Mysidopsis bahia,
Survival, Growth, and
Fecundity Test Method
1007.0, Section 14.11.1,
Summary of Test
Conditions and Test
Acceptability Criteria,
Table 3, Line 18,
Dilution Water, Page
242
Uncontaminated source of natural
seawater, deionized water mixed
with hypersaline brine or artificial
sea salts (HW MARINEMIX®,
FORTY FATHOMS®, GP2 or
equivalent) (available options)
Delete "FORTY
FATHOMS®," from
sentence.
...artificial sea salts (HW MARINEMIX®, GP2 or
equivalent)....
FORTY FATHOMS®, a
commercial product, is no
longer available. Equivalent
commercial products, such as
HW MARINEMIX®, are
acceptable replacements.
Section 15 Sea Urchin,
Fertilization Test
Method, Section
15.6.18.3.8 Consumable
Materials, Table 1,
Footnote 1, Page 301
Stir solutions 1 h to ensure that the
salts dissolve. The salinity of the
initial 840 mL of 100% effluent is
adjusted to 30%oby adding 25.2 g
of dry artificial sea salts (FORTY
FATHOMS®).
Replace "25.2 g of dry
artificial sea salts (FORTY
FATHOMS®)" with "an
appropriate amount of dry
artificial sea salts (e.g.,
HW MARINEMIX®) in
sentence.
.. .by adding an appropriate amount of dry artificial sea
salts (e.g., HW MARINEMIX®).
FORTY FATHOMS®, a
commercial product, is no
longer available. Equivalent
commercial products, such as
HW MARINEMIX®, are
acceptable replacements.
WET Methods Errata Sheet
24
Finalized December 2016

-------
Errata for Methods for Measuring the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms, Third Edition. U.S. Environmental
Protection Agency, Office of Water, Washington DC. EPA 821-R-02-014, October 2002
Text Location
Current Text
Correction Being Made
Corrected Text
Reason For Correction
Section 15 Sea Urchin,
Arbacia punctulata,
Fertilization Test
Method 1008.0, Section
15.6.19.7, Test
Organisms, Sea Urchins,
Arbacia punctulata,
Page 303
To successfully maintain about 25
adult animals for 7 days at a field
site, a screen-partitioned, 40-L
glass aquarium using aerated,
recirculating, clean saline water
(30%o) and a gravel bed filtration
system, is housed within a water
bath, such as FORTY FATHOMS®
or equivalent (15°C).
Replace "FORTY
FATHOMS®" with "HW
MARINEMIX® or
equivalent" in sentence.
... is housed within a water bath, such as HW
MARINEMIX® or equivalent (15°C).
FORTY FATHOMS®, a
commercial product, is no
longer available. Equivalent
commercial products, such as
HW MARINEMIX®, are
acceptable replacements.
Section 15 Sea Urchin,
Arbacia punctulata,
Fertilization Test
Method 1008.0, Section
15.10.1.3.1, Dilution
Water, Page 304
Dilution water may be
uncontaminated natural seawater
(receiving water), HSB prepared
from natural seawater, or artificial
seawater FORTY FATHOMS® or
GP2 sea salts (see Table 2 and
Section 7, Dilution Water).
Replace "FORTY
FATHOMS®" with "HW
MARINEMIX® or
equivalent," in sentence.
... or artificial seawater HW MARINEMIX® or
equivalent, or GP2 sea salts....
FORTY FATHOMS®, a
commercial product, is no
longer available. Equivalent
commercial products, such as
HW MARINEMIX®, are
acceptable replacements.
Consistency with Section 15,
Table 3, Summary of Test
Conditions..., Line 11,
Dilution Water, Page 308.
Section 15 Sea Urchin,
Arbacia punctulata,
Fertilization Test
Method 1008.0, Section
15.10.7.1.2,
Observations During the
Test, Page 306
Temperature, pH, and salinity are
measured at the beginning of the
exposure period in one test
chamber at each concentration and
in the control.
Replace "of the exposure
period ..." with "and end
of each 24-hr exposure
period in at least one test
chamber at each test
concentration and
control."
Temperature, pH, and salinity are measured at the
beginning and end of each 24-hr exposure period in at
least one test chamber at each test concentration and
control.
Clarification.
Section 15, Sea Urchin,
Arbacia punctulata,
Fertilization Test
Method 1008.0, Section
15.10.8.1, Start of the
Test, Page 306
Sufficient test solution is prepared
at each effluent concentration to
provide additional volume for
chemical analyses, at the high,
medium, and low test
concentrations.
Replace "at the high,
medium and low" with "all
test."
Sufficient test solution is prepared at each effluent
concentration to provide additional volume for chemical
analyses at all test concentrations.
Clarification.
WET Methods Errata Sheet
25
Finalized December 2016

-------
Errata for Methods for Measuring the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms, Third Edition. U.S. Environmental
Protection Agency, Office of Water, Washington DC. EPA 821-R-02-014, October 2002
Text Location
Current Text
Correction Being Made
Corrected Text
Reason For Correction
Section 15 Sea Urchin,
Arbacia punctulata,
Fertilization Test
Method 1008.0, Section
15.11.1, Summary of
Test Conditions and Test
Acceptability Criteria,
Table 3, Line 11,
Dilution Water, Page
308
Uncontaminated source of natural
seawater; deionized water mixed
with hypersaline brine or artificial
sea salts (HW MARINEMIX®,
FORTY FATHOMS®, GP2, or
equivalent) (available options)
Delete "FORTY
FATHOMS®," from
sentence.
...artificial sea salts (HW MARINEMIX®, GP2 or
equivalent)....
FORTY FATHOMS®, a
commercial product, is no
longer available. Equivalent
commercial products, such as
HW MARINEMIX®, are
acceptable replacements.
Section 16, Red
Macroalga, Champia
parvula, Sexual
Reproduction Test
Method 1009.0, Section
16.10.1.3.1, Dilution
Water, Page 343
The formula for the enrichment
for natural seawater is listed in
Table 1. Both EDTA and trace
metals have been omitted. This
formula should be used for the 2-
day exposure period, but it is not
critical for the recovery period.
Since natural seawater quality can
vary among laboratories, a more
complete nutrient medium (e.g.,
the addition of EDTA) may result
in faster growth (and therefore
faster cystocarp development)
during the recovery period.
Add text from Line 12,
Dilution Water, Table 3,
Summary of Test
Conditions..., Page 348
30%o salinity natural seawater, or a combination of 50%
of 30%o salinity natural seawater and 50% of 30%o
salinity GP2 artificial seawater (see Section 7, Dilution
Water). The formula for the enrichment for....
Consistency with Section 16,
Table 3, Summary of Test
Conditions, Line 12, Dilution
Water, Page 348
Section 16, Red
Macroalga, Champia
parvula, Sexual
Reproduction Test
Method 1009.2, Section
16.10.6.1.2,
Observations During the
Test, Page 344
Temperature, pH, and salinity are
measured at the end of each 24-hr
exposure period in at least one test
chamber at each test concentration
and in the control.
Add "beginning and"
between "at the" and "end
of." Add "at least"
between "period in" and
"one test."
Temperature, pH, and salinity are measured at the
beginning and end of each 24-hr exposure period in at
least one test chamber at each test concentration and in
the control.
Clarification.
Appendix C, Dunnett's
Procedure, Section 2.5,
Computer Calculations,
Page 408
The program was written in IBM-
PC FORTRAN by Computer
Sciences Corporation, 26 W.
Martin Luther King Drive,
Cincinnati, OH 45268. A
compiled version of the program
can be obtained from EMSL-
Cincinnati by sending a diskette
with a written request.
Replace ".. .from EMSL-
Cincinnati...." with
replacement software
information.
... can be obtained by clicking on "contact us" at
htto://www.eDa.eov/med. Anv software capable of
calculating an analysis of variance (ANOVA) and a
multiple of treatment means with the control mean
(Dunnett's Procedure) can be used.
Computer program is no
longer available from EMSL-
Cincinnati. It can be obtained
by sending a request to EPA
using the link. Any software
capable of completing the
calculations is acceptable.
WET Methods Errata Sheet
26
Finalized December 2016

-------
Errata for Methods for Measuring the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms, Third Edition. U.S. Environmental
Protection Agency, Office of Water, Washington DC. EPA 821-R-02-014, October 2002
Text Location
Current Text
Correction Being Made
Corrected Text
Reason For Correction
Appendix H, Probit
Analysis, Section 2,
Page 436
A compiled, executable version of
the program and supporting
documentation can be obtained
from EMSL-Cincinnati by
sending a written request to
EMSL at 3411 Church Street,
Cincinnati, OH 45244.
Replace ".. .from EMSL-
Cincinnati. ..." with
replacement software
information.
... can be obtained by clicking on "contact us" at
htto://www.eDa.eov/med. Anv software capable of
calculating the EC1 and EC50 (or LCI and LC50) and
the associated 95% confidence intervals using Probit
analysis can be used.
Computer program is no
longer available from EMSL-
Cincinnati. It can be obtained
by sending a request to EPA
using the link. Any software
capable of completing the
calculations is acceptable.
Appendix J, Trimmed
Spearman-Karber
Method, Section 10,
Page 445
A computer program which
estimates the LC50 and associated
95% confidence interval using the
Trimmed Spearman-Karber
Method, can be obtained through
the EMSL, 3411 Church Street,
Cincinnati, OH 45244. The
program can be obtained from
EMSL-Cincinnati by sending a
written request to the above
address.
Replace ".. .through the
EMSL-Cincinnati...."
with replacement software
information.
... can be obtained by clicking on "contact us" at
htto://www.eDa.eov/med. Anv software capable of
calculating the LC50 and associated 95% confidence
interval using the Trimmed Spearman-Karber Method
can be used.
Computer program is no
longer available from EMSL-
Cincinnati. It can be obtained
by sending a request to EPA
using the link. Any software
capable of completing the
calculations is acceptable.
Appendix L, Linear
Interpolation Method,
Section 7.1, Page 458
A compiled, executable version of
the program and supporting
documentation can be obtained by
sending a written request to
EMSL-Cincinnati, 3411 Church
Street, Cincinnati, OH 45244.
Replace "... sending a
written request to EMSL-
Cincinnati. ..." with
replacement software
information.
... can be obtained by clicking on "contact us" at
htto://www.eDa.eov/med. Anv software capable of
calculating the mean and standard deviation of the
bootstrapped ICp and associated 95% confidence
intervals can be used.
Computer program is no
longer available from EMSL-
Cincinnati. It can be obtained
by sending a request to EPA
using the link. Any software
capable of completing the
calculations is acceptable.
Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms, Fifth Edition. U.S. Environmental Protection Agency, Office
of Water, Washington, DC. EPA 821-R-02-012, October 2002. Short title: Acute Freshwater and Marine Manual.
Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms, Fourth Edition. U.S. Environmental Protection Agency,
Office of Water, Washington, DC. EPA 821-R-02-013, October 2002. Short title: Chronic Freshwater Manual.
Methods for Measuring the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms, Third Edition. U.S. Environmental Protection Agency,
Office of Water, Washington DC. EPA 821-R-02-014, October 2002. Short title: Chronic Marine and Estuarine Manual.
WET Methods Errata Sheet
27
Finalized December 2016

-------