United States
Environmental Protection
Agency
Research and Development
Environmental Research
Laboratory
Duluth MN 55804
EPA-600/S3-84-005 Jan. 1984
SEPA Project Summary
Interlaboratory Comparison of
Continuous Flow, Early Life
Stage Testing with
Fathead Minnows
A. E. Lemke
Six laboratories conducted toxicity
experiments according to a supplied
protocol. Also supplied were the chem-
icals to be tested (acenaphthene and
isophorone). Test organisms were fat-
head minnow (Pimephales promelas)
embryos that were raised until 28 days
post hatch. Each investigator weighed
and compared all fish with the controls,
and the various participants provided all
analytical work and other necessities.
Results ranged between 0.049 mg/l
and 0.42 mg/l for the low solubility
acenaphthene and between 1.35 mg/l
and 45.4 mg/l for a more soluble
isophorone. The isophorone results
were strongly correlated inversely to
growth of the controls, which varied
between 0.969 gr and O.018 gr for a
high and low, respectively.
This Project Summary was developed
by EPA's Environmental Research
Laboratory, Duluth, MN, to announce
key findings of the research project that
is fully documented in a separate report
of the same title (see Project Report
ordering information at back).
Introduction
Toxicity testing is an important facet of
environmental protection strategy. One
of the organisms that has been used for a
large a mount of aquatic toxicology testing
is the fathead minnow (Pimephales
promelas) Comparison of previous re-
search has shown that the early life
stages, especially first feeding fry, are
most sensitive to most toxicants. A best-
available-knowledge method was pre-
pared. This report is the result of efforts
by seven government and private sector
laboratories, all with previous knowledge
and experience with the fathead minnow,
to follow the written protocol.
The laboratories were asked to respond
to the protocol before they were selected,
and all questions raised at that time were
answered. After selection, each labora-
tory was supplied the test chemical from
a central identical source. Questions
presented by the labs were answered
during the testing phase but no effort was
made to control the work other than a
request that participants note any and all
problems for inclusion in their reports.
The participants supplied the water used
in the testing and all bioassay and analyt-
ical chemistry work.
Test evaluation consisted of comparing
the geometric mean of the highest test
concentrations, which was statistically
the same as the control, with the lowest
test concentration, which was statistically
different from the control based on the
mean weight of the test organisms after a
28-day post-hatch exposure. In the case
of the acenaphthene tests, such compari-
sons were also made with a solvent
control. The protocol advises the feeding
of newly hatched brine shrimp nauplii
(Anemia salma), and each participant
stated that this food was used One lab
stated that it had an additional source of
natural food, and one other lab had added
a small amount of commercial trout food
after the first week post hatch
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Conclusions
The reported no-effect values for ace-
naphthene varied from a low of 0 049
mg I to a high of 0 42 mg/l, a factor less
than 10 Means of the paired tests were
closer than this For isophorone the low
value was 1 35 mg I and the high was
45 4 mg I, or a 34-fold difference
The less than 10-fold difference is
common in biological results, while the
other 30 plus difference is regarded as
excessive The causal relationship of
these differences appears to be two-fold
Isopnorone results were inversely corre-
lated to the growth of the controls follow-
ing the least squares formula Y = AeBX
where A -- 24 66 and B = -3 087 with an
R" of 37
All participants reported a minimum of
analvtical problems with all tests The
cause of the lack of growth effect on the
acenaphthene therefore must be found
elsewhere In the author's opinion, it was
a result of the limited solubility of ace-
naphthene, which put only limited stress
on all organisms in the exposure concen-
trations
The variation in growth apparently is in
the feeding regime, and several citations
are included which indicate variation in
Anemia used as food One of the partici-
pants also found that the timing of food
presentation appears critical
Recommendations
It is recommended that the protocol be
modified to require a nonexposure test
run with all conditions except toxicant
included, as experience in a specific
procedure is apparently a prerequisite for
satisfactory results and that a growth
level of 0 1 5 gr mean per fish be con-
sidered an absolute minimum in all
exposures If this growth is not accom-
plished the feed and feeding regime
should be changed until it is, prior to
toxicant testing
This Project Summary was authored by A. E. Lemke (also the EPA Project Officer,
see below) who is with the Environmental Research Laboratory, Du/uth, MN
55804
The complete report, entitled "Interlaboratory Comparison of Continuous Flow,
Early Life Stage Testing with Fathead Minnows," (Order No. PB 84-129 493:
Cost $8.50, subject to change) will be available only from
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone 703-487-4650
The EPA Pro/ect Officer can be contacted at.
Environmental Research Laboratory
U.S Environmental Protect/on Agency
Du/uth, MN 55804
-,US GOVERNMENT PRINTING OFFICE 1984-759-015/7287
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Official Business
Penalty for Private Use $300
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