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Analytical Methods
April 10, 1996
MEMORANDUM
SUBJECT: Clarifications Regarding Flexibility in 40 CFR Part 136 Whole Effluent Toxicity (WET) Test Methods
FROM: Tudor T. Davies, Director
Office of Science and Technology
TO: Water Management Division Directors, Regions I-X
Environmental Services Division Directors, Regions I-X
The purpose of this memorandum is to provide clarifications
regarding the flexibility in the regulations promulgated at 60
Fed. Reg. 53529 (Oct. 16, 1995), which establish analytical test
methods for the determination of whole effluent toxicity (WET).
This information is important to the conduct of the WET test
methods, and I ask that you share this memorandum with the States
in your Region approved to administer the National Pollutant
Discharge Elimination System (NPDES) permitting program. In
addition, I would encourage States to make the information
available to their NPDES permittees and appropriate test
analysts.
The WET test methods are codified at 40 CFR part 136,
including three test method manuals which are incorporated by
reference. Because they are incorporated by reference, the test
method manuals themselves constitute Federal regulations. The
test method manuals do not, however, strictly prescribe every
aspect of method conduct, and it is important to think carefully
about how the WET test data will be used when making decisions on
both the selection of the test species as well as the selection
of individual test parameters where flexibility is allowed.
In many instances, the manuals use discretionary terms such
as "may" or "should," with the understanding
that the laboratory analyst must have flexibility to optimize
successful test completion. In other instances, the manuals use
stronger, compulsory terms such as "shall" or
"must" to prescribe procedures necessary for nationwide
standardization (specifically those procedures that assure the
predictability of the methods to provide accurate results). When
situations arise that are not addressed by the manuals, questions
will become apparent -- where the method manuals neither
authorize nor prohibit a procedure in question. In the majority
of these instances, we anticipate that silence in the manual
will indicate flexibility for the individual analyst. The
remainder of this memorandum addresses issues that have already
arisen in the areas of pH, ammonia, temperature, water hardness,
test dilution concentrations, and a definition of
"mean" number of cystocarps in the Champia
parvula test.
1) pH and Ammonia Control
On p. 40 in the freshwater chronic manual (Sections 8.8.6
and 8.8.8) and p. 42 of the marine chronic manual (Sections
8.8.6), the manuals contain some discussion of the influence that
pH and temperature have on ammonia toxicity. In this instance,
the manuals do provide flexibility to the analyst to control
artificial toxicity caused by pH drift provided that the analyst
verifies the source of toxicity is, in fact, artificial. How to
determine this "artificial toxicity" is described in
Section 8.8.8. As the chronic manuals discuss the use of
acids/bases to adjust the pH, likewise the use of CO2 in the
headspace to control the pH is comparable.
The following are example techniques where pH can be
controlled so that ammonia toxicity can be assessed. The pH can
be controlled using appropriate procedures which do not
significantly alter the nature of the effluent. For example,
any procedure which removes ammonia, but does not remove other
toxicants would be allowed. However, treatment with zeolite,
would not routinely be allowed because it removes other
toxicants. Controlling the carbon dioxide (CO2) environment,
however, would be acceptable if carbon dioxide can be delivered
directly to test chambers with airline tubing and a pipette or by
using a complex solenoid system (on demand only). Another
alternative is to maintain a closed carbon dioxide environment,
delivering a solution of CO2 in oxygen to the closed system.
2) Temperature
On p. 53 in the acute manual (Section 9.12.1) and in Tables
11-17, pages 57-70, the manual describes test temperatures for
each acute test. The chronic test methods lists temperatures for
each discrete species test protocol. A concern that has been
raised is that, due to seasonal temperature fluctuations in
receiving waters, the predictive capacity of the test methods
does not reflect actual receiving water effects, and thus the
methods should allow for seasonal variation in test temperatures.
The acute manual does provide protocols for a wide variety of
test species that encompass a broad range of test temperatures
which could approximate the seasonal temperatures found in the
receiving waters. Thus, to accommodate permittee concerns about
the predictive capacity of the standardized test species to
predict toxicity on a seasonal basis, permits may be written to
include different approved test species for different seasons of
the year. In this instance, however, the acute and chronic
manuals do prescribe test temperatures in order to standardize
the methods and limit test variability. Therefore, where
variations in test temperature need to be taken into account,
NPDES permits should include different approved test species,
rather than authorizing seasonal variations in test temperatures
for a given test species.
3) Hardness
For the acute manual, the freshwater chronic manual, and the
marine/estuarine chronic manual, Section 7 in each manual
describes the use of dilution waters. In Appendix A of the acute
manual, and in each specific test Section of the chronic manuals,
the manuals describe how to culture or obtain the test organisms.
Hardness of dilution water, as well as hardness of water used to
culture test organisms, may have an effect on successful
completion of the tests. In some cases, the relative hardness of
the dilution water compared to the organism culture water) may
affect the expression of toxicity in the conduct of the tests,
i.e., the accuracy of the tests at predicting toxicity. In this
regard, the analyst has flexibility in performing the tests to
optimize successful test completion. The type of the dilution
water used in the effluent toxicity tests depends on the
objective of the study. Tests can be conducted in the standard
reconstituted dilution water to assess the absolute toxicity of
the effluent. To evaluate whether or not the toxicity is present
in the receiving water, the test can be conducted with a single
grab sample of receiving water, or the hardness of the dilution
water can be adjusted to match that of the receiving water (while
taking care not to exceed the water hardness tolerance of the
test organisms, which would cause stress to the test organisms,
and affect the toxicity test results). In any case, if the
dilution water is different than the culture water, then a second
set of controls should be tested.
4) Test Dilution Concentrations
The acute manual on p. 47 (Section 9.3.2), freshwater
chronic manual on p. 42 (Section 8.10.2-3), and the marine
manual on p. 45 (Section 8.10.2-3) describes whole effluent
toxicity test dilution concentrations. Although permitting
agencies might assume that a 100% effluent concentration must be
included in the sample series, the manuals do not require this.
The language "such as" is used to show an example of a
concentration series. In some instances, such as marine tests,
using 100% effluent as the highest effluent concentration is not
realistic. In other instances 100% effluent may be unnecessary
if the effluent is not highly variable; or the higher effluent
concentrations are not of concern. The language in the manuals
does, however, recommend bracketing the concentration of concern
(such as the WET permit limit, or the receiving water
concentration of concern). For example, if the effluent
concentration in the receiving water is 12 percent, the dilution
series could be 50 percent, 25 percent, 12.5 percent, 6.25
percent and 3.12 percent effluent.
5) Acceptance Criteria for Champia parvula
The control acceptance criterion for the effluent sexual
reproduction test using Champia parvula is an
average of 10 or more cystocarps for the control treatment (p.
359 of the marine short-term chronic manual, #17). This means
that some replicates may have fewer than 10 cystocarps, and some
more than 10 cystocarps. This is acceptable as long as the
overall mean number of cystocarps per replicate is at least 10
cystocarps.
The analyst may on occasion find that a single control
replicate will have a greatly reduced or enhanced number of
cystocarps relative to the remaining replicates. As with any
toxicity test the analyst may have to determine the single
"odd" replicate is an outlier. If the overall mean
number of cystocarps is at least 10, with the low or high
"odd" replicate excluded, then whether or not a single
outlier is present does not effect the determination of control
acceptability. If the remaining three replicates do not average
10 cystocarps, then the test should be repeated. An outlier,
however, may effect the analyst's conclusions concerning the
level of toxicity in the samples tested along with that
control.
In conclusion, I realize that additional questions may
arise, and I encourage you to contact Teresa Norberg-King in the
Duluth laboratory with these questions. She may be reached at
(218) 529-5163. Over the summer EPA intends to publish a
technical correction notice in the Federal Register for the three
manuals and the part 136 rule. These corrections will not
substantively change the content of the rule or manuals. During
the next few months, EPA will be preparing that notice, and the
list of corrections. Therefore, if you, or your staff are aware
of mistakes or errors, please contact Teresa Norberg-King in
Duluth on or before June 30, 1996. In addition, the Office of
Wastewater Management is working with the Regions and labs to
provide training on the conduct of these methods, as well as
permit writing. For more information on this training effort,
please contact Margaret Heber at (202) 566-1191. If you have any
additional questions, or wish to discuss this further, please
contact me at (202) 566-0430.
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